Methods and compositions for enhancing sensitivity in the analysis of biological-based assays

ABSTRACT

Methods are provided for detecting the binding of a first member to a second member of a ligand pair, comprising the steps of (a) combining a set of first tagged members with a biological sample which may contain one or more second members, under conditions, and for a time sufficient to permit binding of a first member to a second member, wherein said tag is correlative with a particular first member and detectable by non-fluorescent spectrometry, or potentiometry, (b) separating bound first and second members from unbound members, (c) cleaving the tag from the tagged first member, and (d) detecting the tag by non-fluorescent spectrometry, or potentiometry, and therefrom detecting the binding of the first member to the second member.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority according to at least one of 35 U.S.C.§§ 119 and 365 claims priority to, and is a continuation-in-part of,U.S. application Ser. No. 08/787,521, filed Jan. 22, 1997 now abandoned;which claims the benefit of U.S. Provisional Application Ser. No.60/010,436, filed Jan. 23, 1996 and U.S. Provisional Application Ser.No. 60/015,402 filed Mar. 21, 1996, which application is incorporated byreference in its entirety.

TECHNICAL FIELD

The present invention relates generally to methods and compositions foranalyzing nucleic acid molecules, and more specifically, to the use ofspecialized tags and linkers which may be utilized to enhancesensitivity of the analysis of a wide variety of biological-basedassays.

BACKGROUND OF THE INVENTION

Detection and analysis of nucleic acid molecules are among the mostimportant techniques in biology. They are at the heart of molecularbiology and play a rapidly expanding role in the rest of biology.

Generally, following essentially all biochemical reactions, analysisentails some form of detection step. Of especial concern is thedetection of nucleic acid hybridizations and antibody-antigen binding.Ideally, detection should be sensitive and allow processing of multiplesamples. However, current detection techniques are somewhat limited inboth these characteristics.

Hybridization of nucleic acid molecules is generally detected byautoradiography or phosphor image analysis when the hybridization probecontains a radioactive label or by densitometer when the hybridizationprobe contains a label, such as biotin or digoxin, that is recognized byan enzyme-coupled antibody or ligand.

When a radiolabeled probe is used, detection by autoradiography suffersfrom film limitations, such as reciprocity failure and non-linearity.These film limitations can be overcome by detecting the label byphosphor image analysis. However, radiolabels have safety requirements,increasing resource utilization and necessitating specialized equipmentand personnel training. For such reasons, the use of nonradioactivelabels has been increasing in popularity. In such systems, nucleotidescontain a label, such as biotin or digoxin, which can be detected by anantibody or other molecule that is labeled with an enzyme reactive witha chromogenic substrate. Alternatively, fluorescent labels may be used.These systems do not have the safety concerns as described above, butuse components that are often labile and may yield nonspecificreactions, resulting in high background (i.e., low signal-to-noiseratio).

Antibody-antigen binding reactions may be detected by one of severalprocedures. As for nucleic acid hybridization, a label, radioactive ornonradioactive, is typically conjugated to the antibody. The types oflabels are similar: enzyme reacting with a chromogenic substrate,fluorescent, hapten that is detected by a ligand or another antibody,and the like. As in detection of nucleic acid hybridization, similarlimitations are inherent in these detection methods.

The present invention provides novel compositions which may be utilizedin a wide variety of nucleic acid-based, or protein (e.g.,antibody)-based procedures, and further provides other, relatedadvantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides compositions and methodswhich may be utilized to enhance sensitivity and sample numberthroughput in a wide variety of based assays. In particular, based uponthe inventions described herein, many assays that heretofore have takena long period of time to complete may now be performed ten to more thana hundred-fold faster. The methods described herein thus represent adramatic and important improvement over previously available assays.

For example, within one aspect of the invention methods are provided fordetecting the binding of a first member to a second member of a ligandpair, comprising the steps of (a) combining a set of first taggedmembers with a biological sample which may contain one or more secondmembers, under conditions, and for a time sufficient to permit bindingof a first member to a second member, wherein said tag is correlativewith a particular first member and detectable by non-fluorescentspectrometry, or potentiometry, (b) separating bound first and secondmembers from unbound members, (c) cleaving the tag from the tagged firstmember, and (d) detecting the tag by non-fluorescent spectrometry, orpotentiometry, and therefrom detecting the binding of the first memberto the second member.

A wide variety of first and second member pairs may be utilized withinthe context of the present invention, including for example, nucleicacid molecules (e.g., DNA, RNA, nucleic acid analogues such as PNA, orany combination of these), proteins or polypeptides (e.g., an antibodyor antibody fragment (e.g., monoclonal antibody, polyclonal antibody, ora binding partner such as a CDR), oligosaccharides, hormones, organicmolecules and other substrates (e.g., xenobiotics such asglucuronidase--drug molecule), or any other ligand pair. Within variousembodiments of the invention, the first and second members may be thesame type of molecule or of different types. For example, representativefirst member second member ligand pairs include: nucleic acidmolecule/nucleic acid molecule; antibody/nucleic acid molecule;antibody/hormone; antibody/xenobiotic; and antibody/protein.

Preferably, the first member will recognize either a selected secondember specifically (i.e, to the exclusion of other related molecules),or a class of related second member molecules (e.g., a class of relatedreceptors). Preferably the first member will bind to the second memberwith an affinity of at least about 10⁻⁵ /M, and preferably 10⁻⁶ /M, 10⁻⁷/M, 10⁻⁸ /M, 10⁻⁹ /M, or greater than 10⁻¹² /M. The affinity of a firstmolecule for a second molecule can be readily determined by one ofordinary skill in the art (see Scatchard, Ann. N.Y. Acad. Sci.51:660-672, 1949).

Within other related aspects of the invention, methods are provided foranalyzing the pattern of gene expression from a selected biologicalsample, comprising the steps of (a) exposing nucleic acids from abiological sample, (b) combining the exposed nucleic acids with one ormore selected tagged nucleic acid probes, under conditions and for atime sufficient for said probes to hybridize to said nucleic acids,wherein the tag is correlative with a particular nucleic acid probe anddetectable by non-fluorescent spectrometry, or potentiometry, (c)separating hybridized probes from unhybridized probes, (d) cleaving thetag from the tagged fragment, and (e) detecting the tag bynon-fluorescent spectrometry, or potentiometry, and therefromdetermining the patter of gene expression of the biological sample.Within one embodiment, the biological sample may be stimulated with aselected molecule prior to the step of exposing the nucleic acids.Representative examples of "stimulants" include nucleic acid molecules,recombinant gene delivery vehicles, organic molecules, hormones,proteins, inflammatory factors, cytokines, drugs, drug candidates,paracrine and autocrine factors, and the like.

Within the context of the present invention it should be understood that"biological samples" include not only samples obtained from livingorganisms (e.g., mammals, fish, bacteria, parasites, viruses, fungi andthe like) or from the environment (e.g., air, water or solid samples),but biological materials which may be artificially or syntheticallyproduced (e.g., phage libraries, organic molecule libraries, pools ofgenomic clones and the like). Representative examples of biologicalsamples include biological fluids (e.g., blood, semen, cerebral spinalfluid, urine), biological cells (e.g., stem cells, B or T cells, livercells, fibroblasts and the like), and biological tissues.

Within various embodiments of the above-described methods, the nucleicacid probes and or molecules of the present invention may be generatedby, for example, a ligation, cleavage or extension (e.g., PCR) reaction.Within other related aspects the nucleic acid probes or molecules may betagged at their 5'-end, and the so-tagged molecules function asoligonucleotide primers or dideoxynucleotide terminators.

Within other embodiments of the invention, 4, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, 100, 200, 250, 300, 350, 400, 450, orgreater than 500 different and unique tagged molecules may be utilizedwithin a given reaction simultaneously, wherein each tag is unique for aselected nucleic acid fragment, probe, or first or second member, andmay be separately identifed.

Within further embodiments of the invention, the tag(s) may be detectedby fluorometry, mass spectrometry, infrared spectrometry, ultravioletspectrometry, or, potentiostatic amperometry (e.g., utilizingcoulometric or amperometric detectors). Representative examples ofsuitable spectrometric techniques include time-of-flight massspectrometry, quadrupole mass spectrometry, magnetic sector massspectrometry and electric sector mass spectrometry. Specific embodimentsof such techniques include ion-trap mass spectrometry, electrosprayionization mass spectrometry, ion-spray mass spectrometry, liquidionization mass spectrometry, atmospheric pressure ionization massspectrometry, electron ionization mass spectrometry, fast atom bombardionization mass spectrometry, MALDI mass spectrometry, photo-ionizationtime-of-flight mass spectrometry, laser droplet mass spectrometry,MALDI-TOF mass spectrometry, APCI mass spectrometry, nano-spray massspectrometry, nebulised spray ionization mass spectrometry, chemicalionization mass spectrometry, resonance ionization mass spectrometry,secondary ionization mass spectrometry and thermospray massspectrometry.

Within yet other embodiments of the invention, the bound first andsecond members, or exposed nucleic acids, may be separated from unboundmembers or molecules by methods such as gel electrophoresis, capillaryelectrophoresis, micro-channel electrophoresis, HPLC, size exclusionchromatography, filtration, polyacrylamide gel electrophoresis, liquidchromatography, reverse size exclusion chromatography, ion-exchangechromatography, reverse phase liquid chromatography, pulsed-fieldelectrophoresis, field-inversion electrophoresis, dialysis, andfluorescence-activated liquid droplet sorting. Alternatively, either thefirst or second member, or exposed nucleic acids may be bound to a solidsupport (e.g., hollow fibers (Amicon Corporation, Danvers, Mass.), beads(Polysciences, Warrington, Pa.), magnetic beads (Robbin Scientific,Mountain View, Calif.), plates, dishes and flasks (Corning Glass Works,Corning, N.Y.), meshes (Becton Dickinson, Mountain View, Calif.),screens and solid fibers (see Edelman et al., U.S. Pat. No. 3,843,324;see also Kuroda etyal., U.S. Pat. No. 4,416,777), membranes (MilliporeCorp., Bedford, Mass.), and dipsticks). If the first or second member,or exposed nucleic acids are bound to a solid support, within certainembodiments of the invention the methods disclosed herein may furthercomprise the step of washing the solid support of unbound material.

Within other embodiments, the tagged first members may be cleaved by amethods such as chemical, oxidation, reduction, acid-labile, baselabile, enzymatic, electrochemical, heat and photolabile methods. Withinfurther embodiments, the steps of separating, cleaving and detecting maybe performed in a continuous manner (e.g., as a continuous flow), forexample, on a single device which may be automated.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth herein whichdescribe in more detail certain procedures or compositions (e.g.,plasmids, etc.), and are therefore incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the flowchart for the synthesis of pentafluorophenylesters of chemically cleavable mass spectroscopy tags, to liberate tagswith carboxyl amide termini.

FIG. 2 depicts the flowchart for the synthesis of pentafluorophenylesters of chemically cleavable mass spectroscopy tags, to liberate tagswith carboxyl acid termini.

FIGS. 3-6 and 8 depict the flowchart for the synthesis oftetrafluorophenyl esters of a set of 36 photochemically cleavable massspectroscopy tags.

FIG. 7 depicts the flowchart for the synthesis of a set of 36amine-terminated photochemically cleavable mass spectroscopy tags.

FIG. 9 depicts the synthesis of 36 photochemically cleavable massspectroscopy tagged oligonucleotides made from the corresponding set of36 tetrafluorophenyl esters of photochemically cleavable massspectroscopy tag acids.

FIG. 10 depicts the synthesis of 36 photochemically cleavable massspectroscopy tagged oligonucleotides made from the corresponding set of36 amine-terminated photochemically cleavable mass spectroscopy tags.

FIG. 11 illustrates the simultaneous detection of multiple tags by massspectrometry.

FIG. 12 shows the mass spectrogram of the alpha-cyano matrix alone.

FIG. 13 depicts a modularly-constructed tagged nucleic acid fragment.

FIG. 14 is a schematic representation of an array interrogation systemusing Matrix Assisted Laser Desorption Ionization (MALDI) massspectroscopy in accordance with an embodiment of the present invention.

FIGS. 15A and 15B illustrate the preparation of a cleavable tag of thepresent invention.

FIGS. 16A and 16B illustrate the preparation of a cleavable tag of thepresent invention.

FIG. 17 illustrates the preparation of an intermediate compound usefulin the preparation of a cleavable tag of the invention.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention provides tags and linkers that maybe utilized to enhance sensitivity and sample number in a wide varietyof biological-based assays. Described in more detail below arerepresentative tags and linkers that may be utilized, a wide variety ofmethods wherein the tags may be useful, and methods for detecting thetags.

Briefly stated, in one aspect the present invention provides compoundswherein a molecule of interest, or precursor thereto, is linked via alabile bond (or labile bonds) to a tag. Thus, compounds of the inventionmay be viewed as having the general formula:

    T-L-X

wherein T is the tag component, L is the linker component that eitheris, or contains, a labile bond, and X is either the molecule of interest(MOI) component or a functional group component (L_(h)) through whichthe MOI may be joined to T-L. Compounds of the invention may thereforebe represented by the more specific general formulas:

    T-L-MOI and T-L-L.sub.h

For reasons described in detail below, sets of T-L-MOI compounds may bepurposely subjected to conditions that cause the labile bond(s) tobreak, thus releasing a tag moiety from the remainder of the compound.The tag moiety is then characterized by one or more analyticaltechniques, to thereby provide direct information about the structure ofthe tag moiety, and (most importantly) indirect information about theidentity of the corresponding MOI.

As a simple illustrative example of a representative compound of theinvention wherein L is a direct bond, reference is made to the followingstructure (i): ##STR1## In structure (i), T is a nitrogen-containingpolycyclic aromatic moiety bonded to a carbonyl group, X is a MOI (andspecifically a nucleic acid fragment terminating in an amine group), andL is the bond which forms an amide group. The amide bond is labilerelative to the bonds in T because, as recognized in the art, an amidebond may be chemically cleaved (broken) by acid or base conditions whichleave the bonds within the tag component unchanged. Thus, a tag moiety(i.e., the cleavage product that contains T) may be released as shownbelow: ##STR2##

However, the linker L may be more than merely a direct bond, as shown inthe following illustrative example, where reference is made to anotherrepresentative compound of the invention having the structure (ii) shownbelow: ##STR3## It is well-known that compounds having anortho-nitrobenzylamine moiety (see boxed atoms within structure (ii))are photolytically unstable, in that exposure of such compounds toactinic radiation of a specified wavelength will cause selectivecleavage of the benzylamine bond (see bond denoted with heavy line instructure (ii)). Thus, structure (ii) has the same T and MOI groups asstructure (i), however the linker group contains multiple atoms andbonds within which there is a particularly labile bond. Photolysis ofstructure (ii) thus releases a tag moiety (T-containing moiety) from theremainder of the compound, as shown below. ##STR4##

The invention thus provides compounds which, upon exposure toappropriate cleavage conditions, undergo a cleavage reaction so as torelease a tag moiety from the remainder of the compound. Compounds ofthe invention may be described in terms of the tag moiety, the MOI (orprecursor thereto, L_(h)), and the labile bond(s) which join the twogroups together. Alternatively, the compounds of the invention may bedescribed in terms of the components from which they are formed. Thus,the compounds may be described as the reaction product of a tagreactant, a linker reactant and a MOI reactant, as follows.

The tag reactant consists of a chemical handle (T_(h)) and a variablecomponent (T_(vc)), so that the tag reactant is seen to have the generalstructure:

    T.sub.vc -T.sub.h

To illustrate this nomenclature, reference may be made to structure(iii), which shows a tag reactant that may be used to prepare thecompound of structure (ii). The tag reactant having structure (iii)contains a tag variable component and a tag handle, as shown below:##STR5##

In structure (iii), the tag handle (--C(═O)--A) simply provides anavenue for reacting the tag reactant with the linker reactant to form aT-L moiety. The group "A" in structure (iii) indicates that the carboxylgroup is in a chemically active state, so it is ready for coupling withother handles. "A" may be, for example, a hydroxyl group orpentafluorophenoxy, among many other possibilities. The inventionprovides for a large number of possible tag handles which may be bondedto a tag variable component, as discussed in detail below. The tagvariable component is thus a part of "T" in the formula T-L-X, and willalso be part of the tag moiety that forms from the reaction that cleavesL.

As also discussed in detail below, the tag variable component isso-named because, in preparing sets of compounds according to theinvention, it is desired that members of a set have unique variablecomponents, so that the individual members may be distinguished from oneanother by an analytical technique. As one example, the tag variablecomponent of structure (iii) may be one member of the following set,where members of the set may be distinguished by their UV or massspectra: ##STR6##

Likewise, the linker reactant may be described in terms of its chemicalhandles (there are necessarily at least two, each of which may bedesignated as L_(h)) which flank a linker labile component, where thelinker labile component consists of the required labile moiety (L²) andoptional labile moieties (L¹ and L³), where the optional labile moietieseffectively serve to separate L² from the handles L_(h), and therequired labile moiety serves to provide a labile bond within the linkerlabile component. Thus, the linker reactant may be seen to have thegeneral formula:

    L.sub.h -L.sup.1 -L.sup.2 -L.sup.3 -L.sub.h

The nomenclature used to describe the linker reactant may be illustratedin view of structure (iv), which again draws from the compound ofstructure (ii): ##STR7##

As structure (iv) illustrates, atoms may serve in more than onefunctional role. Thus, in structure (iv), the benzyl nitrogen functionsas a chemical handle in allowing the linker reactant to join to the tagreactant via an amide-forming reaction, and subsequently also serves asa necessary part of the structure of the labile moiety L² in that thebenzylic carbon-nitrogen bond is particularly susceptible to photolyticcleavage. Structure (iv) also illustrates that a linker reactant mayhave an L³ group (in this case, a methylene group), although not have anL¹ group. Likewise, linker reactants may have an L¹ group but not an L³group, or may have L¹ and L³ groups, or may have neither of L¹ nor L³groups. In structure (iv), the presence of the group "P" next to thecarbonyl group indicates that the carbonyl group is protected fromreaction. Given this configuration, the activated carboxyl group of thetag reactant (iii) may cleanly react with the amine group of the linkerreactant (iv) to form an amide bond and give a compound of the formulaT-L-L_(h).

The MOI reactant is a suitably reactive form of a molecule of interest.Where the molecule of interest is a nucleic acid fragment, a suitableMOI reactant is a nucleic acid fragment bonded through its 5' hydroxylgroup to a phosphodiester group and then to an alkylene chain thatterminates in an amino group. This amino group may then react with thecarbonyl group of structure (iv), (after, of course, deprotecting thecarbonyl group, and preferably after subsequently activating thecarbonyl group toward reaction with the amine group) to thereby join theMOI to the linker.

When viewed in a chronological order, the invention is seen to take atag reactant (having a chemical tag handle and a tag variablecomponent), a linker reactant (having two chemical linker handles, arequired labile moiety and 0-2 optional labile moieties) and a MOIreactant (having a molecule of interest component and a chemicalmolecule of interest handle) to form I-L-MOI. Thus, to form T-L-MOI,either the tag reactant and the linker reactant are first reactedtogether to provide T-L-L_(h), and then the MOI reactant is reacted withT-L-L_(h) so as to provide T-L-MOI, or else (less preferably) the linkerreactant and the MOI reactant are reacted together first to provideL_(h) -L-MOI, and then L_(h) -L-MOI is reacted with the tag reactant toprovide T-L-MOI. For purposes of convenience, compounds having theformula T-L-MOI will be described in terms of the tag reactant, thelinker reactant and the MOI reactant which may be used to form suchcompounds. Of course, the same compounds of formula T-L-MOI could beprepared by other (typically, more laborious) methods, and still fallwithin the scope of the inventive T-L-MOI compounds.

In any event, the invention provides that a T-L-MOI compound besubjected to cleavage conditions, such that a tag moiety is releasedfrom the remainder of the compound. The tag moiety will comprise atleast the tag variable component, and will typically additionallycomprise some or all of the atoms from the tag handle, some or all ofthe atoms from the linker handle that was used to join the tag reactantto the linker reactant, the optional labile moiety L¹ if this group waspresent in T-L-MOI, and will perhaps contain some part of the requiredlabile moiety L² depending on the precise structure of L² and the natureof the cleavage chemistry. For convenience, the tag moiety may bereferred to as the T-containing moiety because T will typicallyconstitute the major portion (in terms of mass) of the tag moiety.

Given this introduction to one aspect of the present invention, thevarious components T, L and X will be described in detail. Thisdescription begins with the following definitions of certain terms,which will be used hereinafter in describing T, L and X.

As used herein, the term "nucleic acid fragment" means a molecule whichis complementary to a selected target nucleic acid molecule (i.e.,complementary to all or a portion thereof), and may be derived fromnature or synthetically or recombinantly produced, includingnon-naturally occurring molecules, and may be in double or singlestranded form where appropriate; and includes an oligonucleotide (e.g.,DNA or RNA), a primer, a probe, a nucleic acid analog (e.g., PNA), anoligonucleotide which is extended in a 5' to 3' direction by apolymerase, a nucleic acid which is cleaved chemically or enzymatically,a nucleic acid that is terminated with a dideoxy terminator or capped atthe 3' or 5' end with a compound that prevents polymerization at the 5'or 3' end, and combinations thereof. The complementarity of a nucleicacid fragment to a selected target nucleic acid molecule generally meansthe exhibition of at least about 70% specific base pairing throughoutthe length of the fragment. Preferably the nucleic acid fragmentexhibits at least about 80% specific base pairing; and most preferablyat least about 90%. Assays for determining the percent mismatch (andthus the percent specific base pairing) are well known in the art andare based upon the percent mismatch as a function of the Tm whenreferenced to the fully base paired control.

As used herein, the term "alkyl," alone or in combination, refers to asaturated, straight-chain or branched-chain hydrocarbon radicalcontaining from 1 to 10, preferably from 1 to 6 and more preferably from1 to 4, carbon atoms. Examples of such radicals include, but are notlimited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, decyl and the like. Theterm "alkylene" refers to a saturated, straight-chain or branched chainhydrocarbon diradical containing from 1 to 10, preferably from 1 to 6and more preferably from 1 to 4, carbon atoms. Examples of suchdiradicals include, but are not limited to, methylene, ethylene (--CH₂--CH₂ --), propylene, and the like.

The term "alkenyl," alone or in combination, refers to a straight-chainor branched-chain hydrocarbon radical having at least one carbon-carbondouble bond in a total of from 2 to 10, preferably from 2 to 6 and morepreferably from 2 to 4, carbon atoms. Examples of such radicals include,but are not limited to, ethenyl, E- and Z-propenyl, isopropenyl, E- andZ-butenyl, E- and Z-isobutenyl, E- and Z-pentenyl, decenyl and the like.The term "alkenylene" refers to a straight-chain or branched-chainhydrocarbon diradical having at least one carbon-carbon double bond in atotal of from 2 to 10, preferably from 2 to 6 and more preferably from 2to 4, carbon atoms. Examples of such diradicals include, but are notlimited to, methylidene (═CH₂), ethylidene (--CH═CH--), propylidene(--CH₂ --CH═CH--) and the like.

The term "alkynyl," alone or in combination, refers to a straight-chainor branched-chain hydrocarbon radical having at least one carbon-carbontriple bond in a total of from 2 to 10, preferably from 2 to 6 and morepreferably from 2 to 4, carbon atoms. Examples of such radicals include,but are not limited to, ethynyl (acetylenyl), propynyl (propargyl),butynyl, hexynyl, decynyl and the like. The term "alkynylene", alone orin combination, refers to a straight-chain or branched-chain hydrocarbondiradical having at least one carbon-carbon triple bond in a total offrom 2 to 10, preferably from 2 to 6 and more preferably from 2 to 4,carbon atoms. Examples of such radicals include, but are not limited,ethynylene (--C.tbd.C--), propynylene (--CH₂ --C.tbd.C--) and the like.

The term "cycloalkyl," alone or in combination, refers to a saturated,cyclic arrangement of carbon atoms which number from 3 to 8 andpreferably from 3 to 6, carbon atoms. Examples of such cycloalkylradicals include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl and the like. The term "cycloalkylene" refers toa diradical form of a cycloalkyl.

The term "cycloalkenyl," alone or in combination, refers to a cycliccarbocycle containing from 4 to 8, preferably 5 or 6, carbon atoms andone or more double bonds. Examples of such cycloalkenyl radicalsinclude, but are not limited to, cyclopentenyl, cyclohexenyl,cyclopentadienyl and the like. The term "cycloalkenylene" refers to adiradical form of a cycloalkenyl.

The term "aryl" refers to a carbocyclic (consisting entirely of carbonand hydrogen) aromatic group selected from the group consisting ofphenyl, naphthyl, indenyl, indanyl, azulenyl, fluorenyl, andanthracenyl; or a heterocyclic aromatic group selected from the groupconsisting of furyl, thienyl, pyridyl, pyrrolyl, oxazolyly, thiazolyl,imidazolyl, pyrazolyl, 2-pyrazolinyl, pyrazolidinyl, isoxazolyl,isothiazolyl, 1,2,3-oxadiazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl,pyridazinyl, pyrimidinyl. pyrazinyl, 1,3,5-triazinyl, 1,3,5-trithianyl,indolizinyl, indolyl, isoindolyl, 3H-indolyl, indolinyl,benzo[b]furanyl, 2,3-dihydrobenzofuranyl, benzo[b]thiophenyl,1H-indazolyl, benzimidazolyl, benzthiazolyl, purinyl, 4H-quinolizinyl,quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl,quinoxalinyl, 1,8-naphthyridinyl, pteridinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, and phenoxazinyl.

"Aryl" groups, as defined in this application may independently containone to four substituents which are independently selected from the groupconsisting of hydrogen, halogen, hydroxyl, amino, nitro,trifluoromethyl, trifluoromethoxy, alkyl, alkenyl, alkynyl, cyano,carboxy, carboalkoxy, 1,2-dioxyethylene, alkoxy, alkenoxy or alkynoxy,alkylamino, alkenylamino, alkynylamino, aliphatic or aromatic acyl,alkoxy-carbonylamino, alkylsulfonylamino, morpholinocarbonylamino,thiomorpholinocarbonylamino, N-alkyl guanidino, aralkylaminosulfonyl;aralkoxyalkyl; N-aralkoxyurea; N-hydroxylurea; N-alkenylurea;N,N-(alkyl, hydroxyl)urea; heterocyclyl; thioaryloxy-substituted aryl;N,N-(aryl, alkyl)hydrazino; Ar'-substituted sulfonylheterocyclyl;aralkyl-substituted heterocyclyl; cycloalkyl and cycloakenyl-substitutedheterocyclyl; cycloalkyl-fused aryl; aryloxy-substituted alkyl;heterocyclylamino; aliphatic or aromatic acylaminocarbonyl; aliphatic oraromatic acyl-substituted alkenyl; Ar'-substituted aminocarbonyloxy;Ar', Ar'-disubstituted aryl; aliphatic or aromatic acyl-substitutedacyl; cycloalkylcarbonylalkyl; cycloalkyl-substituted amino;aryloxycarbonylalkyl; phosphorodiamidyl acid or ester;

"Ar'" is a carbocyclic or heterocyclic aryl group as defined abovehaving one to three substituents selected from the group consisting ofhydrogen, halogen, hydroxyl, amino, nitro, trifluoromethyl,trifluoromethoxy, alkyl, alkenyl, alkynyl, 1,2-dioxymethylene,1,2-dioxyethylene, alkoxy, alkenoxy, alkynoxy, alkylamino, alkenylaminoor alkynylamino, alkylcarbonyloxy, aliphatic or aromatic acyl,alkylcarbonylamino, alkoxycarbonylamino, alkylsulfonylamino, N-alkyl orN,N-dialkyl urea.

The term "alkoxy," alone or in combination, refers to an alkyl etherradical, wherein the term "alkyl" is as defined above. Examples ofsuitable alkyl ether radicals include, but are not limited to, methoxy,ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy,tert-butoxy and the like.

The term "alkenoxy," alone or in combination, refers to a radical offormula alkenyl-O--, wherein the term "alkenyl" is as defined aboveprovided that the radical is not an enol ether. Examples of suitablealkenoxy radicals include, but are not limited to, allyloxy, E- andZ-3-methyl-2-propenoxy and the like.

The term "alkynyloxy," alone or in combination, refers to a radical offormula alkynyl-O--, wherein the term "alkynyl" is as defined aboveprovided that the radical is not an ynol ether. Examples of suitablealkynoxy radicals include, but are not limited to, propargyloxy,2-butynyloxy and the like.

The term "thioalkoxy" refers to a thioether radical of formula alkyl-S-,wherein alkyl is as defined above.

The term "alkylamino," alone or in combination, refers to a mono- ordi-alkyl-substituted amino radical (i.e., a radical of formulaalkyl-NH-- or (alkyl)₂ -N-), wherein the term "alkyl" is as definedabove. Examples of suitable alkylamino radicals include, but are notlimited to, methylamino, ethylamino, propylamino, isopropylamino,t-butylamino, N,N-diethylamino and the like.

The term "alkenylamino," alone or in combination, refers to a radical offormula alkenyl-NH- or (alkenyl)₂ N-, wherein the term "alkenyl" is asdefined above, provided that the radical is not an enamine. An exampleof such alkenylamino radicals is the allylamino radical.

The term "alkynylamino," alone or in combination, refers to a radical offormula alkynyl--NH-- or (alkynyl)₂ N--, wherein the term "alkynyl" isas defined above, provided that the radical is not an ynamine. Anexample of such alkynylamino radicals is the propargyl amino radical.

The term "amide" refers to either --N(R¹)--C(═O)-- or --C(═O)--N(R¹)--where R¹ is defined herein to include hydrogen as well as other groups.The term "substituted amide" refers to the situation where R¹ is nothydrogen, while the term "unsubstituted amide" refers to the situationwhere R¹ is hydrogen.

The term "aryloxy," alone or in combination, refers to a radical offormula aryl-O--, wherein aryl is as defined above. Examples of aryloxyradicals include, but are not limited to, phenoxy, naphthoxy, pyridyloxyand the like.

The term "arylamino," alone or in combination, refers to a radical offormula aryl-NH--, wherein aryl is as defined above. Examples ofarylamino radicals include, but are not limited to, phenylamino(anilido), naphthylamino, 2-, 3- and 4-pyridylamino and the like.

The term "aryl-fused cycloalkyl," alone or in combination, refers to acycloalkyl radical which shares two adjacent atoms with an aryl radical,wherein the terms "cycloalkyl" and "aryl" are as defined above Anexample of an aryl-fused cycloalkyl radical is the benzofused cyclobutylradical.

The term "alkylcarbonylamino," alone or in combination, refers to aradical of formula alkyl-CONH, wherein the term "alkyl" is as definedabove.

The term "alkoxycarbonylamino," alone or in combination, refers to aradical of formula alkyl-OCONH--, wherein the term "alkyl" is as definedabove.

The term "alkylsulfonylamino," alone or in combination, refers to aradical of formula alkyl-SO₂ NH--, wherein the term "alkyl" is asdefined above.

The term "arylsulfonylamino," alone or in combination, refers to aradical of formula aryl-SO₂ NH--, wherein the term "aryl" is as definedabove.

The term "N-alkylurea," alone or in combination, refers to a radical offormula alkyl--NH--CO--NH--, wherein the term "alkyl" is as definedabove.

The term "N-arylurea," alone or in combination, refers to a radical offormula aryl--NH--CO--NH--, wherein the term "aryl" is as defined above.

The term "halogen" means fluorine, chlorine, bromine and iodine.

The term "hydrocarbon radical" refers to an arrangement of carbon andhydrogen atoms which need only a single hydrogen atom to be anindependent stable molecule. Thus, a hydrocarbon radical has one openvalence site on a carbon atom, through which the hydrocarbon radical maybe bonded to other atom(s). Alkyl, alkenyl, cycloalkyl, etc. areexamples of hydrocarbon radicals.

The term "hydrocarbon diradical" refers to an arrangement of carbon andhydrogen atoms which need two hydrogen atoms in order to be anindependent stable molecule. Thus, a hydrocarbon radical has two openvalence sites on one or two carbon atoms, through which the hydrocarbonradical may be bonded to other atom(s). Alkylene, alkenylene,alkynylene, cycloalkylene, etc. are examples of hydrocarbon diradicals.

The term "hydrocarbyl" refers to any stable arrangement consistingentirely of carbon and hydrogen having a single valence site to which itis bonded to another moiety, and thus includes radicals known as alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl (without heteroatomincorporation into the aryl ring), arylalkyl, alkylaryl and the like.Hydrocarbon radical is another name for hydrocarbyl.

The term "hydrocarbylene" refers to any stable arrangement consistingentirely of carbon and hydrogen having two valence sites to which it isbonded to other moieties, and thus includes alkylene, alkenylene,alkynylene, cycloalkylene, cycloalkenylene, arylene (without heteroatomincorporation into the arylene ring), arylalkylene, alkylarylene and thelike. Hydrocarbon diradical is another name for hydrocarbylene.

The term "hydrocarbyl-O-hydrocarbylene" refers to a hydrocarbyl groupbonded to an oxygen atom, where the oxygen atom is likewise bonded to ahydrocarbylene group at one of the two valence sites at which thehydrocarbylene group is bonded to other moieties. The terms"hydrocarbyl-S-hydrocarbylene", "hydrocarbyl-NH-hydrocarbylene" and"hydrocarbyl-amide-hydrocarbylene" have equivalent meanings, whereoxygen has been replaced with sulfur, --NH-- or an amide group,respectively.

The term N-(hydrocarbyl)hydrocarbylene refers to a hydrocarbylene groupwherein one of the two valence sites is bonded to a nitrogen atom, andthat nitrogen atom is simultaneously bonded to a hydrogen and ahydrocarbyl group. The term N,N-di(hydrocarbyl)hydrocarbylene refers toa hydrocarbylene group wherein one of the two valence sites is bonded toa nitrogen atom, and that nitrogen atom is simultaneously bonded to twohydrocarbyl groups.

The term "hydrocarbylacyl-hydrocarbylene" refers to a hydrocarbyl groupbonded through an acyl (--C(═O)--) group to one of the two valence sitesof a hydrocarbylene group.

The terms "heterocyclylhydrocarbyl" and "heterocylyl" refer to a stable,cyclic arrangement of atoms which include carbon atoms and up to fouratoms (referred to as heteroatoms) selected from oxygen, nitrogen,phosphorus and sulfur. The cyclic arrangement may be in the form of amonocyclic ring of 3-7 atoms, or a bicyclic ring of 8-11 atoms. Therings may be saturated or unsaturated (including aromatic rings), andmay optionally be benzofused. Nitrogen and sulfur atoms in the ring maybe in any oxidized form, including the quaternized form of nitrogen. Aheterocyclylhydrocarbyl may be attached at any endocyclic carbon orheteroatom which results in the creation of a stable structure.Preferred heterocyclylhydrocarbyls include 5-7 membered monocyclicheterocycles containing one or two nitrogen heteroatoms.

A substituted heterocyclylhydrocarbyl refers to aheterocyclylhydrocarbyl as defined above, wherein at least one ring atomthereof is bonded to an indicated substituent which extends off of thering.

In referring to hydrocarbyl and hydrocarbylene groups, the term"derivatives of any of the foregoing wherein one or more hydrogens isreplaced with an equal number of fluorides" refers to molecules thatcontain carbon, hydrogen and fluoride atoms, but no other atoms.

The term "activated ester" is an ester that contains a "leaving group"which is readily displaceable by a nucleophile, such as an amine, andalcohol or a thiol nucleophile. Such leaving groups are well known andinclude, without limitation, N-hydroxysuccinimide,N-hydroxybenzotriazole, halogen (halides), alkoxy includingtetrafluorophenolates, thioalkoxy and the like. The term "protectedester" refers to an ester group that is masked or otherwise unreactive.See, e.g., Greene, "Protecting Groups In Organic Solutions."

In view of the above definitions, other chemical terms used throughoutthis application can be easily understood by those of skill in the art.Terms may be used alone or in any combination thereof. The preferred andmore preferred chain lengths of the radicals apply to all suchcombinations.

Genation of Tagged Nuclic Acid Fragments

As noted above, one aspect of the present invention provides a generalscheme for DNA sequencing which allows the use of more than 16 tags ineach lane; with continuous detection, the tags can be detected and thesequence read as the size separation is occurring, just as withconventional fluorescence-based sequencing. This scheme is applicable toany of the DNA sequencing techniques based on size separation of taggedmolecules. Suitable tags and linkers for use within the presentinvention, as well as methods for sequencing nucleic acids, arediscussed in more detail below.

1. Tags

"Tag", as used herein, generally refers to a chemical moiety which isused to uniquely identify a "molecule of interest", and morespecifically refers to the tag variable component as well as whatevermay be bonded most closely to it in any of the tag reactant, tagcomponent and tag moiety.

A tag which is useful in the present invention possesses severalattributes:

1) It is capable of being distinguished from all other tags. Thisdiscrimination from other chemical moieties can be based on thechromatographic behavior of the tag (particularly after the cleavagereaction), its spectroscopic or potentiometric properties, or somecombination thereof. Spectroscopic methods by which tags are usefullydistinguished include mass spectroscopy (MS), infrared (IR), ultraviolet(UV), and fluorescence, where MS, IR and UV are preferred, and MS mostpreferred spectroscopic methods. Potentiometric amperometry is apreferred potentiometric method.

2) The tag is capable of being detected when present at 10⁻²² to 10⁻⁶mole.

3) The tag possesses a chemical handle through which it can be attachedto the MOI which the tag is intended to uniquely identify. Theattachment may be made directly to the MOI, or indirectly through a"linker" group.

4) The tag is chemically stable toward all manipulations to which it issubjected, including attachment and cleavage from the MOI, and anymanipulations of the MOI while the tag is attached to it.

5) The tag does not significantly interfere with the manipulationsperformed on the MOI while the tag is attached to it. For instance, ifthe tag is attached to an oligonucleotide, the tag must notsignificantly interfere with any hybridization or enzymatic reactions(e.g., PCR sequencing reactions) performed on the oligonucleotide.Similarly, if the tag is attached to an antibody, it must notsignificantly interfere with antigen recognition by the antibody.

A tag moiety which is intended to be detected by a certain spectroscopicor potentiometric method should possess properties which enhance thesensitivity and specificity of detection by that method. Typically, thetag moiety will have those properties because they have been designedinto the tag variable component, which will typically constitute themajor portion of the tag moiety. In the following discussion, the use ofthe word "tag" typically refers to the tag moiety (i.e., the cleavageproduct that contains the tag variable component), however can also beconsidered to refer to the tag variable component itself because that isthe portion of the tag moiety which is typically responsible forproviding the uniquely detectable properties. In compounds of theformula T-L-X, the "T" portion will contain the tag variable component.Where the tag variable component has been designed to be characterizedby, e.g., mass spectrometry, the "T" portion of T-L-X may be referred toas T^(ms). Likewise, the cleavage product from T-L-X that contains T maybe referred to as the T^(ms) -containing moiety. The followingspectroscopic and potentiometric methods may be used to characterizeT^(ms) -containing moieties.

a. Characteristics of MS Tags

Where a tag is analyzable by mass spectrometry (i.e., is a MS-readabletag, also referred to herein as a MS tag or "T^(ms) -containingmoiety"), the essential feature of the tag is that it is able to beionized. It is thus a preferred element in the design of MS-readabletags to incorporate therein a chemical functionality which can carry apositive or negative charge under conditions of ionization in the MS.This feature confers improved efficiency of ion formation and greateroverall sensitivity of detection, particularly in electrosprayionization. The chemical functionality that supports an ionized chargemay derive from T^(ms) or L or both. Factors that can increase therelative sensitivity of an analyte being detected by mass spectrometryare discussed in, e.g., Sunner, J., et al., Anal. Chem. 60:1300-1307(1988).

A preferred functionality to facilitate the carrying of a negativecharge is an organic acid, such as phenolic hydroxyl, carboxylic acid,phosphonate, phosphate, tetrazole, sulfonyl urea, perfluoro alcohol andsulfonic acid.

Preferred functionality to facilitate the carrying of a positive chargeunder ionization conditions are aliphatic or aromatic amines. Examplesof amine functional groups which give enhanced detectability of MS tagsinclude quaternary amines (i.e., amines that have four bonds, each tocarbon atoms, see Aebersold, U.S. Pat. No. 5,240,859) and tertiaryamines (i.e., amines that have three bonds, each to carbon atoms, whichincludes C═N--C groups such as are present in pyridine, see Hess et al.,Anal. Biochem. 224:373, 1995; Bures et al., Anal. Biochem. 224:364,1995). Hindered tertiary amines are particularly preferred. Tertiary andquaternary amines may be alkyl or aryl. A T^(ms) -containing moiety mustbear at least one ionizable species, but may possess more than oneionizable species. The preferred charge state is a single ionizedspecies per tag. Accordingly, it is preferred that each T^(ms)-containing moiety (and each tag variable component) contain only asingle hindered amine or organic acid group.

Suitable amine-containing radicals that may form part of the T^(ms)-containing moiety include the following: ##STR8##

The identification of a tag by mass spectrometry is preferably basedupon its molecular mass to charge ratio (m/z). The preferred molecularmass range of MS tags is from about 100 to 2,000 daltons, and preferablythe T^(ms) -containing moiety has a mass of at least about 250 daltons,more preferably at least about 300 daltons, and still more preferably atleast about 350 daltons. It is generally difficult for massspectrometers to distinguish among moieties having parent ions belowabout 200-250 daltons (depending on the precise instrument), and thuspreferred T^(ms) -containing moieties of the invention have masses abovethat range.

As explained above, the T^(ms) -containing moiety may contain atomsother than those present in the tag variable component, and indeed otherthan present in T^(ms) itself. Accordingly, the mass of T^(ms) itselfmay be less than about 250 daltons, so long as the T^(ms) -containingmoiety has a mass of at least about 250 daltons. Thus, the mass ofT^(ms) may range from 15 (i.e., a methyl radical) to about 10,000daltons, and preferably ranges from 100 to about 5,000 daltons, and morepreferably ranges from about 200 to about 1,000 daltons.

It is relatively difficult to distinguish tags by mass spectrometry whenthose tags incorporate atoms that have more than one isotope insignificant abundance. Accordingly, preferred T groups which areintended for mass spectroscopic identification (T^(ms) groups), containcarbon, at least one of hydrogen and fluoride, and optional atomsselected from oxygen, nitrogen, sulfur, phosphorus and iodine. Whileother atoms may be present in the T^(ms), their presence can renderanalysis of the mass spectral data somewhat more difficult. Preferably,the T^(ms) groups have only carbon, nitrogen and oxygen atoms, inaddition to hydrogen and/or fluoride.

Fluoride is an optional yet preferred atom to have in a T^(ms) group. Incomparison to hydrogen, fluoride is, of course, much heavier. Thus, thepresence of fluoride atoms rather than hydrogen atoms leads to T^(ms)groups of higher mass, thereby allowing the T^(ms) group to reach andexceed a mass of greater than 250 daltons, which is desirable asexplained above. In addition, the replacement of hydrogen with fluorideconfers greater volatility on the T^(ms) -containing moiety, and greatervolatility of the analyte enhances sensitivity when mass spectrometry isbeing used as the detection method.

The molecular formula of T^(ms) falls within the scope of C₁₋₅₀₀ N₀₋₁₀₀O₀₋₁₀₀ S₀₋₁₀ P₀₋₁₀ H.sub.α F.sub.β I.sub.δ wherein the sum of α, β and δis sufficient to satisfy the otherwise unsatisfied valencies of the C,N, O, S and P atoms. The designation C₁₋₅₀₀ N₀₋₁₀₀ O₀₋₁₀₀ S₀₋₁₀ P₀₋₁₀H.sub.α F.sub.β I.sub.δ means that T^(ms) contains at least one, and maycontain any number from 1 to 500 carbon atoms, in addition to optionallycontaining as many as 100 nitrogen atoms ("N₀₋ " means that T^(ms) neednot contain any nitrogen atoms), and as many as 100 oxygen atoms, and asmany as 10 sulfur atoms and as many as 10 phosphorus atoms. The symbolsα, β and δ represent the number of hydrogen, fluoride and iodide atomsin T^(ms), where any two of these numbers may be zero, and where the sumof these numbers equals the total of the otherwise unsatisfied valenciesof the C, N, O, S and P atoms. Preferably, T^(ms) has a molecularformula that falls within the scope of C₁₋₅₀ N₀₋₁₀ O₀₋₁₀ H.sub.α F.sub.βwhere the sum of ax and D equals the number of hydrogen and fluorideatoms, respectively, present in the moiety.

b. Characteristics of IR Tags

There are two primary forms of IR detection of organic chemical groups:Raman scattering IR and absorption IR. Raman scattering IR spectra andabsorption IR spectra are complementary spectroscopic methods. Ingeneral, Raman excitation depends on bond polarizability changes whereasIR absorption depends on bond dipole moment changes. Weak IR absorptionlines become strong Raman lines and vice versa. Wavenumber is thecharacteristic unit for IR spectra. There are 3 spectral regions for IRtags which have separate applications: near IR at 12500 to 4000 cm⁻¹,mid IR at 4000 to 600 cm⁻¹, far IR at 600 to 30 cm⁻¹. For the usesdescribed herein where a compound is to serve as a tag to identify anMOI, probe or primer, the mid spectral regions would be preferred. Forexample, the carbonyl stretch (1850 to 1750 cm⁻¹) would be measured forcarboxylic acids, carboxylic esters and amides, and alkyl and arylcarbonates, carbamates and ketones. N--H bending (1750 to 160 cm⁻¹)would be used to identify amines, ammonium ions, and amides. At 1400 to1250 cm⁻¹, R--OH bending is detected as well as the C--N stretch inamides. Aromatic substitution patterns are detected at 900 to 690 cm⁻¹(C--H bending, N--H bending for ArNH₂). Saturated C--H, olefins,aromatic rings, double and triple bonds, esters, acetals, ketals,ammonium salts, N--O compounds such as oximes, nitro, N-oxides, andnitrates, azo, hydrazones, quinones, carboxylic acids, amides, andlactams all possess vibrational infrared correlation data (see Pretschet al., Spectral Data for Structure Determination of Organic Compounds,Springer-Verlag, New York, 1989). Preferred compounds would include anaromatic nitrile which exhibits a very strong nitrile stretchingvibration at 2230 to 2210 cm⁻¹. Other useful types of compounds arearomatic alkynes which have a strong stretching vibration that givesrise to a sharp absorption band between 2140 and 2100 cm⁻¹. A thirdcompound type is the aromatic azides which exhibit an intense absorptionband in the 2160 to 2120 cm⁻¹ region. Thiocyanates are representative ofcompounds that have a strong absorption at 2275 to 2263 cm⁻¹.

c. Characteristics of UV Tags

A compilation of organic chromophore types and their respectiveUV-visible properties is given in Scott (Interpretation of the UVSpectra of Natural Products, Permagon Press, New York, 1962). Achromophore is an atom or group of atoms or electrons that areresponsible for the particular light absorption. Empirical rules existfor the π to π* maxima in conjugated systems (see Pretsch et al.,Spectral Data for Structure Determination of Organic Compounds, p. B65and B70, Springer-Verlag, New York, 1989). Preferred compounds (withconjugated systems) would possess n to π* and π to π* transitions. Suchcompounds are exemplified by Acid Violet 7, Acridine Orange, AcridineYellow G, Brilliant Blue G, Congo Red, Crystal Violet, Malachite Greenoxalate, Metanil Yellow, Methylene Blue, Methyl Orange, Methyl Violet B,Naphtol Green B, Oil Blue N, Oil Red O, 4-phenylazophenol, Safranie O,Solvent Green 3, and Sudan Orange G, all of which are commerciallyavailable (Aldrich, Milwaukee, Wis.). Other suitable compounds arelisted in, e.g., Jane, I., et al., J. Chrom. 323:191-225 (1985).

d. Characteristic of a Fluorescent Tag

Fluorescent probes are identified and quantitated most directly by theirabsorption and fluorescence emission wavelengths and intensities.Emission spectra (fluorescence and phosphorescence) are much moresensitive and permit more specific measurements than absorption spectra.Other photophysical characteristics such as excited-state lifetime andfluorescence anisotropy are less widely used. The most generally usefulintensity parameters are the molar extinction coefficient (ε) forabsorption and the quantum yield (QY) for fluorescence. The value of δis specified at a single wavelength (usually the absorption maximum ofthe probe), whereas QY is a measure of the total photon emission overthe entire fluorescence spectral profile. A narrow optical bandwidth(<20 nm) is usually used for fluorescence excitation (via absorption),whereas the fluorescence detection bandwidth is much more variable,ranging from full spectrum for maximal sensitivity to narrow band (˜20nm) for maximal resolution. Fluorescence intensity per probe molecule isproportional to the product of ε and QY. The range of these parametersamong fluorophores of current practical importance is approximately10,000 to 100,000 cm⁻¹ M⁻¹ for ε and 0.1 to 1.0 for QY. Compounds thatcan serve as fluorescent tags are as follows: fluorescein, rhodamine,lambda blue 470, lambda green, lambda red 664, lambda red 665, acridineorange, and propidium iodide, which are commercially available fromLambda Fluorescence Co. (Pleasant Gap, Pa.). Fluorescent compounds suchas nile red, Texas Red, lissamine™, BODIPY™ s are available fromMolecular Probes (Eugene, Oreg.).

e. Characteristics of Potentiometric Tags

The principle of electrochemical detection (ECD) is based on oxidationor reduction of compounds which at certain applied voltages, electronsare either donated or accepted thus producing a current which can bemeasured. When certain compounds are subjected to a potentialdifference, the molecules undergo a molecular rearrangement at theworking electrodes' surface with the loss (oxidation) or gain(reduction) of electrons, such compounds are said to be electronic andundergo electrochemical reactions. EC detectors apply a voltage at anelectrode surface over which the HPLC eluent flows. Electroactivecompounds eluting from the column either donate electrons (oxidize) oracquire electrons (reduce) generating a current peak in real time.Importantly the amount of current generated depends on both theconcentration of the analyte and the voltage applied, with each compoundhaving a specific voltage at which it begins to oxidize or reduce. Thecurrently most popular electrochemical detector is the amperometricdetector in which the potential is kept constant and the currentproduced from the electrochemical reaction is then measured. This typeof spectrometry is currently called "potentiostatic amperometry".Commercial amperometers are available from ESA, Inc., Chelmford, Mass.

When the efficiency of detection is 100%, the specialized detectors aretermed "coulometric". Coulometric detectors are sensitive which have anumber of practical advantages with regard to selectivity andsensitivity which make these types of detectors useful in an array. Incoulometric detectors, for a given concentration of analyte, the signalcurrent is plotted as a function of the applied potential (voltage) tothe working electrode. The resultant sigmoidal graph is called thecurrent-voltage curve or hydrodynamic voltammagram (HDV). The HDV allowsthe best choice of applied potential to the working electrode thatpermits one to maximize the observed signal. A major advantage of ECD isits inherent sensitivity with current levels of detection in thesubfemtomole range.

Numerous chemicals and compounds are electrochemically active includingmany biochemicals, pharmaceuticals and pesticides. Chromatographicallycoeluting compounds can be effectively resolved even if their half-wavepotentials (the potential at half signal maximum) differ by only 30-60mV.

Recently developed coulometric sensors provide selectivity,identification and resolution of co-eluting compounds when used asdetectors in liquid chromatography based separations. Therefore, thesearrayed detectors add another set of separations accomplished in thedetector itself. Current instruments possess 16 channels which are inprinciple limited only by the rate at which data can be acquired. Thenumber of compounds which can be resolved on the EC array ischromatographically limited (i.e., plate count limited). However, if twoor more compounds that chromatographically co-elute have a difference inhalf wave potentials of 30-60 mV, the array is able to distinguish thecompounds. The ability of a compound to be electrochemically activerelies on the possession of an EC active group (i.e., --OH, --O--, N,--S).

Compounds which have been successfully detected using coulometricdetectors include 5-hydroxytryptamine, 3-methoxy-4-hydroxyphenyl-glycol,homogentisic acid, dopamine, metanephrine, 3-hydroxykynureninr,acetominophen, 3-hydroxytryptophol, 5-hydroxyindoleacetic acid,octanesulfonic acid, phenol, o-cresol, pyrogallol, 2-nitrophenol,4-nitrophenol, 2,4-dinitrophenol, 4,6-dinitrocresol,3-methyl-2-nitrophenol, 2,4-dichlorophenol, 2,6-dichlorophenol,2,4,5-trichlorophenol, 4-chloro-3-methylphenol, 5-methylphenol,4-methyl-2-nitrophenol, 2-hydroxyaniline, 4-hydroxyaniline,1,2-phenylenediamine, benzocatechin, buturon chlortholuron, diuron,isoproturon, linuron, methobromuron, metoxuron, monolinuron, monuron,methionine, tryptophan, tyrosine, 4-aminobenzoic acid, 4-hydroxybenzoicacid, 4-hydroxycoumaric acid, 7-methoxycoumarin, apigenin baicalein,caffeic acid, catechin, centaurein, chlorogenic acid, daidzein,datiscetin, diosmetin, epicatechin gallate, epigallo catechin, epigallocatechin gallate, eugenol, eupatorin, ferulic acid, fisetin, galangin,gallic acid, gardenin, genistein, gentisic acid, hesperidin, irigenin,kaemferol, leucoyanidin, luteolin, mangostin, morin, myricetin,naringin, narirutin, pelargondin, peonidin, phloretin, pratensein,protocatechuic acid, rhamnetin, quercetin, sakuranetin, scutellarein,scopoletin, syringaldehyde, syringic acid, tangeritin, troxerutin,umbelliferone, vanillic acid, 1,3-dimethyl tetrahydroisoquinoline,6-hydroxydopamine, r-salsolinol, N-methyl-r-salsolinol,tetrahydroisoquinoline, amitriptyline, apomorphine, capsaicin,chlordiazepoxide, chlorpromazine, daunorubicin, desipramine, doxepin,fluoxetine, flurazepam, imipramine, isoproterenol, methoxamine,morphine, morphine-3-glucuronide, nortriptyline, oxazepam,phenylephrine, trimipramine, ascorbic acid, N-acetyl serotonin,3,4-dihydroxybenzylamine, 3,4-dihydroxymandelic acid (DOMA),3,4-dihydroxyphenylacetic acid (DOPAC), 3,4-dihydroxyphenylalanine(L-DOPA), 3,4-dihydroxyphenylglycol (DHPG), 3-hydroxyanthranilic acid,2-hydroxyphenylacetic acid (2HPAC), 4-hydroxybenzoic acid (4HBAC),5-hydroxyindole-3-acetic acid (5HIAA), 3-hydroxykynurenine,3-hydroxymandelic acid, 3-hydroxy-4-methoxyphenylethylamine,4-hydroxyphenylacetic acid (4HPAC), 4-hydroxyphenyllactic acid (4HPLA),5-hydroxytryptophan (5HTP), 5-hydroxytryptophol (5HTOL),5-hydroxytryptamine (5HT), 5-hydroxytryptamine sulfate,3-methoxy-4-hydroxyphenylglycol (MHPG), 5-methoxytryptamine,5-methoxytryptophan, 5-methoxytryptophol, 3-methoxytyramine (3MT),3-methoxytyrosine (3-OM-DOPA), 5-methylcysteine, 3-methylguanine,bufotenin, dopamine dopamine-3-glucuronide, dopamine-3-sulfate,dopamine-4-sulfate, epinephrine, epinine, folic acid, glutathione(reduced), guanine, guanosine, homogentisic acid (HGA), homovanillicacid (HVA), homovanillyl alcohol (HVOL), homoveratic acid, hva sulfate,hypoxanthine, indole, indole-3-acetic acid, indole-3-lactic acid,kynurenine, melatonin, metanephrine, N-methyltryptamine,N-methyltyramine, N,N-dimethyltryptamine, N,N-dimethyltyramine,norepinephrine, normetanephrine, octopamine, pyridoxal, pyridoxalphosphate, pyridoxamine, synephrine, tryptophol, tryptamine, tyramine,uric acid, vanillylmandelic acid (vma), xanthine and xanthosine. Othersuitable compounds are set forth in, e.g., Jane, I., et al. J. Chrom.323:191-225 (1985) and Musch, G., et al., J. Chrom. 348:97-110 (1985).These compounds can be incorporated into compounds of formula T-L-X bymethods known in the art. For example, compounds having a carboxylicacid group may be reacted with amine, hydroxyl, etc. to form amide,ester and other linkages between T and L.

In addition to the above properties, and regardless of the intendeddetection method, it is preferred that the tag have a modular chemicalstructure. This aids in the construction of large numbers ofstructurally related tags using the techniques of combinatorialchemistry. For example, the T^(ms) group desirably has severalproperties. It desirably contains a functional group which supports asingle ionized charge state when the T^(ms) -containing moiety issubjected to mass spectrometry (more simply referred to as a "mass specsensitivity enhancer" group, or MSSE). Also, it desirably can serve asone member in a family of T^(ms) -containing moieties, where members ofthe family each have a different mass/charge ratio, however haveapproximately the same sensitivity in the mass spectrometer. Thus, themembers of the family desirably have the same MSSE. In order to allowthe creation of families of compounds, it has been found convenient togenerate tag reactants via a modular synthesis scheme, so that the tagcomponents themselves may be viewed as comprising modules.

In a preferred modular approach to the structure of the T^(ms) group,T^(ms) has the formula

    T.sup.2 -(J-T.sup.3 -).sub.n -

wherein T² is an organic moiety formed from carbon and one or more ofhydrogen, fluoride, iodide, oxygen, nitrogen, sulfur and phosphorus,having a mass range of 15 to 500 daltons; T³ is an organic moiety formedfrom carbon and one or more of hydrogen, fluoride, iodide, oxygen,nitrogen, sulfur and phosphorus, having a mass range of 50 to 1000daltons; J is a direct bond or a functional group such as amide, ester,amine, sulfide, ether, thioester, disulfide, thioether, urea, thiourea,carbamate, thiocarbamate, Schiff base, reduced Schiff base, imine,oxime, hydrazone, phosphate, phosphonate, phosphoramide, phosphonamide,sulfonate, sulfonamide or carbon-carbon bond; and n is an integerranging from 1 to 50, such that when n is greater than 1, each T³ and Jis independently selected.

The modular structure T² -(J-T³)_(n) - provides a convenient entry tofamilies of T-L-X compounds, where each member of the family has adifferent T group. For instance, when T is T^(ms), and each familymember desirably has the same MSSE, one of the T³ groups can providethat MSSE structure. In order to provide variability between members ofa family in terms of the mass of T^(ms), the T² group may be variedamong family members. For instance, one family member may have T²=methyl, while another has T² =ethyl, and another has T² =propyl, etc.

In order to provide "gross" or large jumps in mass, a T³ group may bedesigned which adds significant (e.g., one or several hundreds) of massunits to T-L-X. Such a T³ group may be referred to as a molecular weightrange adjuster group("WRA"). A WRA is quite useful if one is workingwith a single set of T² groups, which will have masses extending over alimited range. A single set of T² groups may be used to create T^(ms)groups having a wide range of mass simply by incorporating one or moreWRA T³ groups into the T^(ms). Thus, using a simple example, if a set ofT² groups affords a mass range of 250-340 daltons for the T^(ms), theaddition of a single WRA, having, as an exemplary number 100 dalton, asa T³ group provides access to the mass range of 350-440 daltons whileusing the same set of T² groups. Similarly, the addition of two 100dalton MWA groups (each as a T³ group) provides access to the mass rangeof 450-540 daltons, where this incremental addition of WRA groups can becontinued to provide access to a very large mass range for the T^(ms)group. Preferred compounds of the formula T² -(J-T³ -)_(n) -L-X have theformula R_(VWC) -(R_(WRA))_(w) -R_(MSSE) -L-X where VWC is a "T² "group, and each of the WRA and MSSE groups are "T³ " groups. Thisstructure is illustrated in FIG. 12, and represents one modular approachto the preparation of T^(ms).

In the formula T² -(J-T³ -)_(n) -, T² and T³ are preferably selectedfrom hydrocarbyl, hydrocarbyl-O-hydrocarbylene,hydrocarbyl-S-hydrocarbylene, hydrocarbyl-NH-hydrocarbylene,hydrocarbyl-amide-hydrocarbylene, N-(hydrocarbyl)hydrocarbylene,N,N-di(hydrocarbyl)hydrocarbylene, hydrocarbylacylhydrocarbylene,heterocyclylhydrocarbyl wherein the heteroatom(s) are selected fromoxygen, nitrogen, sulfur and phosphorus, substitutedheterocyclylhydrocarbyl wherein the heteroatom(s) are selected fromoxygen, nitrogen, sulfur and phosphorus and the substituents areselected from hydrocarbyl, hydrocarbyl-O-hydrocarbylene,hydrocarbyl-NH-hydrocarbylene, hydrocarbyl-S-hydrocarbylene,N-(hydrocarbyl)hydrocarbylene, N,N-di(hydrocarbyl)hydrocarbylene andhydrocarbylacyl-hydrocarbylene. In addition, T² and/or T³ may be aderivative of any of the previously listed potential T² /T³ groups, suchthat one or more hydrogens are replaced fluorides.

Also regarding the formula T² -(J-T³ -)_(n) -, a preferred T³ has theformula -G(R²)--, wherein G is C₁₋₆ alkylene chain having a single R²substituent. Thus, if G is ethylene (--CH₂ --CH₂ --) either one of thetwo ethylene carbons may have a R² substituent, and R² is selected fromalkyl, alkenyl, alkynyl, cycloalkyl, aryl-fused cycloalkyl,cycloalkenyl, aryl, aralkyl, aryl-substituted alkenyl or alkynyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted cycloalkyl,biaryl, alkoxy, alkenoxy, alkynoxy, aralkoxy, aryl-substituted alkenoxyor alkynoxy, alkylamino, alkenylamino or alkynylamino, aryl-substitutedalkylamino, aryl-substituted alkenylamino or alkynylamino, aryloxy,arylamino, N-alkylurea-substituted alkyl, N-arylurea-substituted alkyl,alkylcarbonylamino-substituted alkyl, aminocarbonyl-substituted alkyl,heterocyclyl, heterocyclyl-substituted alkyl, heterocyclyl-substitutedamino, carboxyalkyl substituted aralkyl, oxocarbocyclyl-fused aryl andheterocyclylalkyl; cycloalkenyl, aryl-substituted alkyl and, aralkyl,hydroxy-substituted alkyl, alkoxy-substituted alkyl,aralkoxy-substituted alkyl, alkoxy-substituted alkyl,aralkoxy-substituted alkyl, amino-substituted alkyl, (aryl-substitutedalkyloxycarbonylamino)-substituted alkyl, thiol-substituted alkyl,alkylsulfonyl-substituted alkyl, (hydroxy-substitutedalkylthio)-substituted alkyl, thioalkoxy-substituted alkyl,hydrocarbylacylamino-substituted alkyl,heterocyclylacylamino-substituted alkyl,hydrocarbyl-substituted-heterocyclylacylamino-substituted alkyl,alkylsulfonylamino-substituted alkyl, arylsulfonylamino-substitutedalkyl, morpholino-alkyl, thiomorpholino-alkyl, morpholinocarbonyl-substituted alkyl, thiomorpholinocarbonyl-substituted alkyl,[N-(alkyl, alkenyl or alkynyl)- or N,N-[dialkyl, dialkenyl, dialkynyl or(alkyl, alkenyl)-amino]carbonyl-substituted alkyl,heterocyclylaminocarbonyl, heterocylylalkyleneaminocarbonyl,heterocyclylaminocarbonyl-substituted alkyl,heterocylylalkyleneaminocarbonyl-substituted alkyl,N,N-[dialkyl]alkyleneaminocarbonyl,N,N-[dialkyl]alkyleneaminocarbonyl-substituted alkyl, alkyl-substitutedheterocyclylcarbonyl, alkyl-substituted heterocyclylcarbonyl-alkyl,carboxyl-substituted alkyl, dialkylamino-substituted acylaminoalkyl andamino acid side chains selected from arginine, asparagine, glutamine,S-methyl cysteine, methionine and corresponding sulfoxide and sulfonederivatives thereof, glycine, leucine, isoleucine, allo-isoleucine,tert-leucine, norleucine, phenylalanine, tyrosine, tryptophan, proline,alanine, ornithine, histidine, glutamine, valine, threonine, serine,aspartic acid, beta-cyanoalanine, and allothreonine; alynyl andheterocyclylcarbonyl, aminocarbonyl, amido, mono- ordialkylaminocarbonyl, mono- or diarylaminocarbonyl,alkylarylaminocarbonyl, diarylaminocarbonyl, mono- ordiacylaminocarbonyl, aromatic or aliphatic acyl, alkyl optionallysubstituted by substituents selected from amino, carboxy, hydroxy,mercapto, mono- or dialkylamino, mono- or diarylamino, alkylarylamino,diarylamino, mono- or diacylamino, alkoxy, alkenoxy, aryloxy,thioalkoxy, thioalkenoxy, thioalkynoxy, thioaryloxy and heterocyclyl.

A preferred compound of the formula T² -(J-T³ -)_(n) -L-X has thestructure: ##STR9## wherein G is (CH₂)₁₋₆ such that a hydrogen on oneand only one of the CH₂ groups represented by a single "G" is replacedwith-(CH₂)_(c) -Amide-T⁴ ; T² and T⁴ are organic moieties of the formulaC₁₋₂₅ N₀₋₉ O₀₋₉ H.sub.α F.sub.β such that the sum of α and β issufficient to satisfy the otherwise unsatisfied valencies of the C, N,and O atoms; amide is ##STR10## R¹ is hydrogen or C₁₋₁₀ alkyl; c is aninteger ranging from 0 to 4; and n is an integer ranging from 1 to 50such that when n is greater than 1, G, c, Amide, R¹ and T⁴ areindependently selected.

In a further preferred embodiment, a compound of the formula T² -(J-T³-)_(n) -L-X has the structure: ##STR11##

wherein T⁵ is an organic moiety of the formula C_(i-25) N₀₋₉ O₀₋₉H.sub.α F.sub.β such that the sum of α and β is sufficient to satisfythe otherwise unsatisfied valencies of the C, N, and O atoms; and T⁵includes a tertiary or quaternary amine or an organic acid; m is aninteger ranging from 0-49, and T², T⁴, R¹, L and X have been previouslydefined.

Another preferred compound having the formula T² -(J-T³ -)_(n) -L-X hasthe particular structure: ##STR12## wherein T⁵ is an organic moiety ofthe formula C₁₋₂₅ N₀₋₉ O₀₋₉ H.sub.αF.sub.β such that the sum of α and βis sufficient to satisfy the otherwise unsatisfied valencies of the C,N, and O atoms; and T⁵ includes a tertiary or quaternary amine or anorganic acid; m is an integer ranging from 0-49, and T², T⁴, c, R¹,"Amide", L and X have been previously defined.

In the above structures that have a T⁵ group, -Amide-T⁵ is preferablyone of the following, which are conveniently made by reacting organicacids with free amino groups extending from "G": ##STR13##

Where the above compounds have a T⁵ group, and the "G" group has a freecarboxyl group (or reactive equivalent thereof), then the following arepreferred -Amide-T⁵ group, which may conveniently be prepared byreacting the appropriate organic amine with a free carboxyl groupextending from a "G" group: ##STR14##

In three preferred embodiments of the invention, T-L-MOI has thestructure: ##STR15## or the structure: ##STR16## or the structure:##STR17## wherein T² and T⁴ are organic moieties of the formula C₁₋₂₅N₀₋₉ O₀₋₉ S₀₋₃ P₀₋₃ H.sub.α F.sub.β I.sub.δ such that the sum of α, βand δ is sufficient to satisfy the otherwise unsatisfied valencies ofthe C, N, O, S and P atoms; G is (CH₂)₁₋₆ wherein one and only onehydrogen on the CH₂ groups represented by each G is replaced with--(CH₂)_(c) -Amide-T⁴ ; Amide is ##STR18## R¹ is hydrogen or C₁₋₁₀alkyl; c is an integer ranging from 0 to 4; "C₂ -C₁₀ " represents ahydrocarbylene group having from 2 to 10 carbon atoms, "ODN-3'-OH"represents a nucleic acid fragment having a terminal 3' hydroxyl group(i.e., a nucleic acid fragment joined to (C₁ -C₁₀) at other than the 3'end of the nucleic acid fragment); and n is an integer ranging from 1 to50 such that when n is greater than 1, then G, c, Amide, R¹ and T⁴ areindependently selected. Preferably there are not three heteroatomsbonded to a single carbon atom.

In structures as set forth above that contain a T² -C(═O)--N(R¹)--group, this group may be formed by reacting an amine of the formulaHN(R¹)-- with an organic acid selected from the following, which areexemplary only and do not constitute an exhaustive list of potentialorganic acids: Formic acid, Acetic acid, Propiolic acid, Propionic acid,Fluoroacetic acid, 2-Butynoic acid, Cyclopropanecarboxylic acid, Butyricacid, Methoxyacetic acid, Difluoroacetic acid, 4-Pentynoic acid,Cyclobutanecarboxylic acid, 3,3-Dimethylacrylic acid, Valeric acid,N,N-Dimethylglycine, N-Formyl-Gly-OH, Ethoxyacetic acid,(Methylthio)acetic acid, Pyrrole-2-carboxylic acid, 3-Furoic acid,Isoxazole-5-carboxylic acid, trans-3-Hexenoic acid, Trifluoroaceticacid, Hexanoic acid, Ac-Gly-OH, 2-Hydroxy-2-methylbutyric acid, Benzoicacid, Nicotinic acid, 2-Pyrazinecarboxylic acid,l-Methyl-2-pyrrolecarboxylic acid, 2-Cyclopentene-1-acetic acid,Cyclopentylacetic acid, (S)-(-)-2-Pyrrolidone-5-carboxylic acid,N-Methyl-1-proline, Heptanoic acid, Ac-b-Ala-OH,2-Ethyl-2-hydroxybutyric acid, 2-(2-Methoxyethoxy)acetic acid, p-Toluicacid, 6-Methylnicotinic acid, 5-Methyl-2-pyrazinecarboxylic acid,2,5-Dimethylpyrrole-3-carboxylic acid, 4-Fluorobenzoic acid,3,5-Dimethylisoxazole-4-carboxylic acid, 3-Cyclopentylpropionic acid,Octanoic acid, N,N-Dimethylsuccinamic acid, Phenylpropiolic acid,Cinnamic acid, 4-Ethylbenzoic acid, p-Anisic acid,1,2,5-Trimethylpyrrole-3-carboxylic acid, 3-Fluoro-4-methylbenzoic acid,Ac-DL-Propargylglycine, 3-(Trifluoromethyl)butyric acid,1-Piperidinepropionic acid, N-Acetylproline, 3,5-Difluorobenzoic acid,Ac-L-Val-OH, Indole-2-carboxylic acid, 2-Benzofurancarboxylic acid,Benzotriazole-5-carboxylic acid, 4-n-Propylbenzoic acid,3-Dimethylaminobenzoic acid, 4-Ethoxybenzoic acid, 4-(Methylthio)benzoicacid, N-(2-Furoyl)glycine, 2-(Methylthio)nicotinic acid,3-Fluoro-4-methoxybenzoic acid, Tfa-Gly-OH, 2-Napthoic acid, Quinaldicacid, Ac-L-Ile-OH, 3-Methylindene-2-carboxylic acid,2-Quinoxalinecarboxylic acid, 1-Methylindole-2-carboxylic acid,2,3,6-Trifluorobenzoic acid, N-Formyl-L-Met-OH,2-[2-(2-Methoxyethoxy)ethoxy]acetic acid, 4-n-Butylbenzoic acid,N-Benzoylglycine, 5-Fluoroindole-2-carboxylic acid, 4-n-Propoxybenzoicacid, 4-Acetyl-3,5-dimethyl-2-pyrrolecarboxylic acid,3,5-Dimethoxybenzoic acid, 2,6-Dimethoxynicotinic acid,Cyclohexanepentanoic acid, 2-Naphthylacetic acid,4-(1H-Pyrrol-1-yl)benzoic acid, Indole-3-propionic acid,m-Trifluoromethylbenzoic acid, 5-Methoxyindole-2-carboxylic acid,4-Pentylbenzoic acid, Bz-b-Ala-OH, 4-Diethylaminobenzoic acid,4-n-Butoxybenzoic acid, 3-Methyl-5-CF3-isoxazole-4-carboxylic acid,(3,4-Dimethoxyphenyl)acetic acid, 4-Biphenylcarboxylic acid,Pivaloyl-Pro-OH, Octanoyl-Gly-OH, (2-Naphthoxy)acetic acid,Indole-3-butyric acid, 4-(Trifluoromethyl)phenylacetic acid,5-Methoxyindole-3-acetic acid, 4-(Trifluoromethoxy)benzoic acid,Ac-L-Phe-OH, 4-Pentyloxybenzoic acid, Z-Gly-OH,4-Carboxy-N-(fur-2-ylmethyl)pyrrolidin-2-one, 3,4-Diethoxybenzoic acid,2,4-Dimethyl-5-CO₂ Et-pyrrole-3-carboxylic acid,N-(2-Fluorophenyl)succinamic acid, 3,4,5-Trimethoxybenzoic acid,N-Phenylanthranilic acid, 3-Phenoxybenzoic acid, Nonanoyl-Gly-OH,2-Phenoxypyridine-3-carboxylic acid,2,5-Dimethyl-1-phenylpyrrole-3-carboxylic acid,trans-4-(Trifluoromethyl)cinnamic acid,(5-Methyl-2-phenyloxazol-4-yl)acetic acid, 4-(2-Cyclohexenyloxy)benzoicacid, 5-Methoxy-2-methylindole-3-acetic acid, trans-4-Cotininecarboxylicacid, Bz-5-Aminovaleric acid, 4-Hexyloxybenzoic acid,N-(3-Methoxyphenyl)succinamic acid, Z-Sar-OH,4-(3,4-Dimethoxyphenyl)butyric acid, Ac-o-Fluoro-DL-Phe-OH,N-(4-Fluorophenyl)glutaramic acid, 4'-Ethyl-4-biphenylcarboxylic acid,1,2,3,4-Tetrahydroacridinecarboxylic acid, 3-Phenoxyphenylacetic acid,N-(2,4-Difluorophenyl)succinamic acid, N-Decanoyl-Gly-OH,(+)-6-Methoxy-a-methyl-2-naphthaleneacetic acid,3-(Trifluoromethoxy)cinnamic acid, N-Formyl-DL-Trp-OH,(R)-(+)-a-Methoxy-a-(trifluoromethyl)phenylacetic acid, Bz-DL-Leu-OH,4-(Trifluoromethoxy)phenoxyacetic acid, 4-Heptyloxybenzoic acid,2,3,4-Trimethoxycinnamic acid, 2,6-Dimethoxybenzoyl-Gly-OH,3-(3,4,5-Trimethoxyphenyl)propionic acid,2,3,4,5,6-Pentafluorophenoxyacetic acid,N-(2,4-Difluorophenyl)glutaramic acid, N-Undecanoyl-Gly-OH,2-(4-Fluorobenzoyl)benzoic acid, 5-Trifluoromethoxyindole-2-carboxylicacid, N-(2,4-Difluorophenyl)diglycolamic acid, Ac-L-Trp-OH,Tfa-L-Phenylglycine-OH, 3-Iodobenzoic acid,3-(4-n-Pentylbenzoyl)propionic acid, 2-Phenyl-4-quinolinecarboxylicacid, 4-Octyloxybenzoic acid, Bz-L-Met-OH, 3,4,5-Triethoxybenzoic acid,N-Lauroyl-Gly-OH, 3,5-Bis(trifluoromethyl)benzoic acid,Ac-5-Methyl-DL-Trp-OH, 2-Iodophenylacetic acid, 3-Iodo-4-methylbenzoicacid, 3-(4-n-Hexylbenzoyl)propionic acid, N--Hexanoyl-L-Phe-OH,4-Nonyloxybenzoic acid, 4'-(Trifluoromethyl)-2-biphenylcarboxylic acid,Bz-L-Phe-OH, N-Tridecanoyl-Gly-OH, 3,5-Bis(trifluoromethyl)phenylaceticacid, 3-(4-n-Heptylbenzoyl)propionic acid, N-Hepytanoyl-L-Phe-OH,4-Decyloxybenzoic acid, N-(α,α,α-trifluoro-m-tolyl)anthranilic acid,Niflumic acid, 4-(2-Hydroxyhexafluoroisopropyl)benzoic acid,N-Myristoyl-Gly-OH, 3-(4-n-Octylbenzoyl)propionic acid,N-Octanoyl-L-Phe-OH, 4-Undecyloxybenzoic acid,3-(3,4,5-Trimethoxyphenyl)propionyl-Gly-OH, 8-Iodonaphthoic acid,N-Pentadecanoyl-Gly-OH, 4-Dodecyloxybenzoic acid, N-Palmitoyl-Gly-OH,and N-Stearoyl-Gly-OH. These organic acids are available from one ormore of Advanced ChemTech, Louisville, Ky.; Bachem Bioscience Inc.,Torrance, Calif.; Calbiochem-Novabiochem Corp., San Diego, Calif.;Farchan Laboratories Inc., Gainesville Fla.; Lancaster Synthesis,Windham N.H.; and MayBridge Chemical Company (c/o Ryan Scientific),Columbia, S.C. The catalogs from these companies use the abreviationswhich are used above to identify the acids.

f. Combinatorial Chemistry as a Means for Preparing Tags

Combinatorial chemistry is a type of synthetic strategy which leads tothe production of large chemical libraries (see, for example, PCTApplication Publication No. WO 94/08051). These combinatorial librariescan be used as tags for the identification of molecules of interest(MOIs). Combinatorial chemistry may be defined as the systematic andrepetitive, covalent connection of a set of different "building blocks"of varying structures to each other to yield a large array of diversemolecular entities. Building blocks can take many forms, both naturallyoccurring and synthetic, such as nucleophiles, electrophiles, dienes,alkylating or acylating agents, diamines, nucleotides, amino acids,sugars, lipids, organic monomers, synthons, and combinations of theabove. Chemical reactions used to connect the building blocks mayinvolve alkylation, acylation, oxidation, reduction, hydrolysis,substitution, elimination, addition, cyclization, condensation, and thelike. This process can produce libraries of compounds which areoligomeric, non-oligomeric, or combinations thereof. If oligomeric, thecompounds can be branched, unbranched, or cyclic. Examples of oligomericstructures which can be prepared by combinatorial methods includeoligopeptides, oligonucleotides, oligosaccharides, polylipids,polyesters, polyamides, polyurethanes, polyureas, polyethers,poly(phosphorus derivatives), e.g., phosphates, phosphonates,phosphoramides, phosphonamides, phosphites, phosphinamides, etc., andpoly(sulfur derivatives), e.g., sulfones, sulfonates, sulfites,sulfonamides, sulfenamides, etc.

One common type of oligomeric combinatorial library is the peptidecombinatorial library. Recent innovations in peptide chemistry andmolecular biology have enabled libraries consisting of tens to hundredsof millions of different peptide sequences to be prepared and used. Suchlibraries can be divided into three broad categories. One category oflibraries involves the chemical synthesis of soluble non-support-boundpeptide libraries (e.g., Houghten et al., Nature 354:84, 1991). A secondcategory involves the chemical synthesis of support-bound peptidelibraries, presented on solid supports such as plastic pins, resinbeads, or cotton (Geysen et al., Mol. Immunol. 23:709, 1986; Lam et al.,Nature 354:82, 1991; Eichler and Houghten, Biochemistry 32:11035, 1993).In these first two categories, the building blocks are typically L-aminoacids, D-amino acids, unnatural amino acids, or some mixture orcombination thereof. A third category uses molecular biology approachesto prepare peptides or proteins on the surface of filamentous phageparticles or plasmids (Scott and Craig, Curr. Opinion Biotech. 5:40,1994). Soluble, nonsupport-bound peptide libraries appear to be suitablefor a number of applications, including use as tags. The availablerepertoire of chemical diversities in peptide libraries can be expandedby steps such as permethylation (Ostresh et al., Proc. Natl. Acad. Sci.,USA 91:11138, 1994).

Numerous variants of peptide combinatorial libraries are possible inwhich the peptide backbone is modified, and/or the amide bonds have beenreplaced by mimetic groups. Amide mimetic groups which may be usedinclude ureas, urethanes, and carbonylmethylene groups. Restructuringthe backbone such that sidechains emanate from the amide nitrogens ofeach amino acid, rather than the alpha-carbons, gives libraries ofcompounds known as peptoids (Simon et al., Proc. Natl. Acad. Sci., USA89:9367, 1992).

Another common type of oligomeric combinatorial library is theoligonucleotide combinatorial library, where the building blocks aresome form of naturally occurring or unnatural nucleotide orpolysaccharide derivatives, including where various organic andinorganic groups may substitute for the phosphate linkage, and nitrogenor sulfur may substitute for oxygen in an ether linkage (Schneider etal., Biochem. 34:9599, 1995; Freier et al., J. Med. Chem. 38:344, 1995;Frank, J. Biotechnology 41:259, 1995; Schneider et al., Published PCT WO942052; Ecker et al., Nucleic Acids Res. 21:1853, 1993).

More recently, the combinatorial production of collections ofnon-oligomeric, small molecule compounds has been described (DeWitt etal., Proc. Natl. Acad. Sci., USA 90:690, 1993; Bunin et al., Proc. Natl.Acad. Sci., USA 91:4708, 1994). Structures suitable for elaboration intosmall-molecule libraries encompass a wide variety of organic molecules,for example heterocyclics, aromatics, alicyclics, aliphatics, steroids,antibiotics, enzyme inhibitors, ligands, hormones, drugs, alkaloids,opioids, terpenes, porphyrins, toxins, catalysts, as well ascombinations thereof

g. Specific Methods for Combinatorial Synthesis of Tags

Two methods for the preparation and use of a diverse set ofamine-containing MS tags are outlined below. In both methods, solidphase synthesis is employed to enable simultaneous parallel synthesis ofa large number of tagged linkers, using the techniques of combinatorialchemistry. In the first method, the eventual cleavage of the tag fromthe oligonucleotide results in liberation of a carboxyl amide. In thesecond method, cleavage of the tag produces a carboxylic acid. Thechemical components and linking elements used in these methods areabbreviated as follows:

R=resin

FMOC=fluorenylmethoxycarbonyl protecting group

All=allyl protecting group

CO₂ H=carboxylic acid group

CONH₂ =carboxylic amide group

NH₂ =amino group

OH=hydroxyl group

CONH=amide linkage

COO=ester linkage

NH₂ --Rink-CO₂ H=4-[(α-amino)-2,4-dimethoxybenzyl]-phenoxybutyric acid(Rink linker)

OH-1MeO--CO₂ H=(4-hydroxymethyl)phenoxybutyric acid

OH-2MeO--CO₂ H=(4-hydroxymethyl-3-methoxy)phenoxyacetic acid

NH₂ -A-COOH=amino acid with aliphatic or aromatic amine functionality inside chain

X1 . . . Xn-COOH=set of n diverse carboxylic acids with unique molecularweights

oligo1 . . . oligo(n)=set of n oligonucleotides

HBTU=O-benzotriazol-1-yl-N,N,N',N'-tetramethyluroniumhexafluorophosphate

The sequence of steps in Method 1 is as follows:

    ______________________________________                                        OH-2MeO-CONH-R                                                                     ↓  FMOC-NH-Rink-CO.sub.2 H; couple (e.g, HBTU)                    FMOC-NH-Rink-COO-2MeO-CONH-R                                                       ↓  piperidine (remove FMOC)                                       NH.sub.2 -Rink-COO-2MeO-CONH-R                                                     ↓  FMOC-NH-A-COOH; couple (e.g, HBTU)                             FMOC-NH-A-CONH-Rink-COO-2MeO-CONH-R                                                ↓  piperidine (remove FMOC)                                       NH.sub.2 -A-CONH-Rink-COO-2MeO-CONH-R                                              ↓  divide into n aliquots                                           ↓↓↓↓↓ couple to n different acids X1                    . . . Xn-COOH                                                  X1 . . .Xn-CONH-A-CONH-Rink-COO-2Me-CONH-R                                         ↓↓↓↓↓                                                Cleave tagged linkers from resin with 1% TFA                   X1 . . . Xn-CONH-A-CONH-Rink-CO.sub.2 H                                            ↓↓↓↓↓                                                couple to n oligos (oligo 1 . . . oligo(n))                       (e.g., via Pfp esters)                                                     X1 . . . Xn-CONH-A-CONH-Rink-CONH-oligo1 . . . oligo(n)                            ↓  pool tagged oligos                                               ↓ perform sequencing reaction                                          ↓ separate different length fragments from                              sequencing reaction (e.g., via HPLC or CB)                                   ↓ cleave tags from linkers with 25%-100% TFA                         X1 . . . Xn-CONH-A-CONH                                                            ↓                                                                 analyze by mass spectrometry                                                  ______________________________________                                    

The sequence of steps in Method 2 is as follows:

    ______________________________________                                        OH-1MeO-CO.sub.2 -All                                                              ↓  FMOC-NH-A-CO.sub.2 H; couple (e.g., HBTU)                      FMOC-NH-A-COO-1MeO-CO.sub.2 -All                                                   ↓  Palladium (remove Allyl)                                       FMOC-NH-A-COO-1MeO-CO.sub.2 H                                                      ↓  OH-2MeO-CONH-R; couple (e.g., HBTU)                            FMOC-NH-A-COO-1MeO-COO-2MeO-CONH-R                                                 ↓  piperidine (remove FMOC)                                       NH.sub.2 -A-COO-1MeO-COO-2MeO-CONH-R                                               ↓  divide into n aliquots                                           ↓↓↓↓↓ couple to n different acids X1                    . . . Xn-CO.sub.2 H                                            X1 . . . Xn-CONH-A-COO-1MeO-COO-2MeO-CONH-R                                        ↓↓↓↓↓                                                cleave tagged linkers from resin with 1% TFA                   X1 . . . Xn-CONH-A-COO-1MeO-CO.sub.2 H                                             ↓↓↓↓↓                                                couple to n oligos (oligo1 . . . oligo(n))                        (e.g., via Pfp esters)                                                     X1 . . . Xn-CONH-A-COO-1MeO-CONH-oligo1 . . . oligo(n)                             ↓  pool tagged oligos                                               ↓ perform sequencing reaction                                          ↓ separate different length fragments from                              sequencing reaction (e.g., via HPLC or CE)                                   ↓ cleave tags from linkers with 25-100% TFA                          X1 . . . Xn-CONH-A-CO.sub.2 H                                                      ↓                                                                 analyze by mass spectrometry                                                  ______________________________________                                    

2. Linkers

A "linker" component (or L), as used herein, means either a directcovalent bond or an organic chemical group which is used to connect a"tag" (or T) to a "molecule of interest" (or MOI) through covalentchemical bonds. In addition, the direct bond itself, or one or morebonds within the linker component is cleavable under conditions whichallows T to be released (in other words, cleaved) from the remainder ofthe T-L-X compound (including the MOI component). The tag variablecomponent which is present within T should be stable to the cleavageconditions. Preferably, the cleavage can be accomplished rapidly; withina few minutes and preferably within about 15 seconds or less.

In general, a linker is used to connect each of a large set of tags toeach of a similarly large set of MOIs. Typically, a single tag-linkercombination is attached to each MOI (to give various T-L-MOI), but insome cases, more than one tag-linker combination may be attached to eachindividual MOI (to give various (T-L)n-MOI). In another embodiment ofthe present invention, two or more tags are bonded to a single linkerthrough multiple, independent sites on the linker, and this multipletag-linker combination is then bonded to an individual MOI (to givevarious (T)n-L-MOI).

After various manipulations of the set of tagged MOIs, special chemicaland/or physical conditions are used to cleave one or more covalent bondsin the linker, resulting in the liberation of the tags from the MOIs.The cleavable bond(s) may or may not be some of the same bonds that wereformed when the tag, linker, and MOI were connected together. The designof the linker will, in large part, determine the conditions under whichcleavage may be accomplished. Accordingly, linkers may be identified bythe cleavage conditions they are particularly susceptible too. When alinker is photolabile (i.e., prone to cleavage by exposure to actinicradiation), the linker may be given the designation L_(h)υ. Likewise,the designations L^(acid), L^(base), L.sup.[O], L.sup.[R], L^(enz),L^(elc), L.sup.Δ and L^(ss) may be used to refer to linkers that areparticularly susceptible to cleavage by acid, base, chemical oxidation,chemical reduction, the catalytic activity of an enzyme (more simply"enzyme"), electrochemical oxidation or reduction, elevated temperature("thermal") and thiol exchange, respectively.

Certain types of linker are labile to a single type of cleavagecondition, whereas others are labile to several types of cleavageconditions. In addition, in linkers which are capable of bondingmultiple tags (to give (T)n-L-MOI type structures), each of thetag-bonding sites may be labile to different cleavage conditions. Forexample, in a linker having two tags bonded to it, one of the tags maybe labile only to base, and the other labile only to photolysis.

A linker which is useful in the present invention possesses severalattributes:

1) The linker possesses a chemical handle (L_(h)) through which it canbe attached to an MOI.

2) The linker possesses a second, separate chemical handle (L_(h))through which the tag is attached to the linker. If multiple tags areattached to a single linker ((T)n-L-MOI type structures), then aseparate handle exists for each tag.

3) The linker is stable toward all manipulations to which it issubjected, with the exception of the conditions which allow cleavagesuch that a T-containing moiety is released from the remainder of thecompound, including the MOI. Thus, the linker is stable duringattachment of the tag to the linker, attachment of the linker to theMOI, and any manipulations of the MOI while the tag and linker (T-L) areattached to it.

4) The linker does not significantly interfere with the manipulationsperformed on the MOI while the T-L is attached to it. For instance, ifthe T-L is attached to an oligonucleotide, the T-L must notsignificantly interfere with any hybridization or enzymatic reactions(e.g., PCR) performed on the oligonucleotide. Similarly, if the T-L isattached to an antibody, it must not significantly interfere withantigen recognition by the antibody.

5) Cleavage of the tag from the remainder of the compound occurs in ahighly controlled manner, using physical or chemical processes that donot adversely affect the detectability of the tag.

For any given linker, it is preferred that the linker be attachable to awide variety of MOIs, and that a wide variety of tags be attachable tothe linker. Such flexibility is advantageous because it allows a libraryof T-L conjugates, once prepared, to be used with several different setsof MOIs.

As explained above, a preferred linker has the formula

    L.sub.h -L.sup.1 L.sup.2 -L.sup.3 -L.sub.h

wherein each L_(h) is a reactive handle that can be used to link thelinker to a tag reactant and a molecule of interest reactant. L² is anessential part of the linker, because L² imparts lability to the linker.L¹ and L³ are optional groups which effectively serve to separate L²from the handles L_(h).

L¹ (which, by definition, is nearer to T than is L³), serves to separateT from the required labile moiety L². This separation may be useful whenthe cleavage reaction generates particularly reactive species (e.g.,free radicals) which may cause random changes in the structure of theT-containing moiety. As the cleavage site is further separated from theT-containing moiety, there is a reduced likelihood that reactive speciesformed at the cleavage site will disrupt the structure of theT-containing moiety. Also, as the atoms in L¹ will typically be presentin the T-containing moiety, these L¹ atoms may impart a desirablequality to the T-containing moiety. For example, where the T-containingmoiety is a T^(ms) -containing moiety, and a hindered amine is desirablypresent as part of the structure of the T^(ms) -containing moiety (toserve, e.g., as a MSSE), the hindered amine may be present in L¹ labilemoiety.

In other instances, L¹ and/or L³ may be present in a linker componentmerely because the commercial supplier of a linker chooses to sell thelinker in a form having such a L¹ and/or L³ group. In such an instance,there is no harm in using linkers having L¹ and/or L³ groups, (so longas these group do not inhibit the cleavage reaction) even though theymay not contribute any particular performance advantage to the compoundsthat incorporate them. Thus, the present invention allows for L¹ and/orL³ groups to be present in the linker component.

L¹ and/or L³ groups may be a direct bond (in which case the group iseffectively not present), a hydrocarbylene group (e.g., alkylene,arylene, cycloalkylene, etc.), --O-hydrocarbylene (e.g., --O--CH₂ --,O--CH₂ CH(CH₃)--, etc.) or hydrocarbylene-(O-hydrocarbylene)_(w) -wherein w is an integer ranging from 1 to about 10 (e.g., --CH₂--O--Ar--, --CH₂ --(O--CH₂ CH₂)₄ --, etc.).

With the advent of solid phase synthesis, a great body of literature hasdeveloped regarding linkers that are labile to specific reactionconditions. In typical solid phase synthesis, a solid support is bondedthrough a labile linker to a reactive site, and a molecule to besynthesized is generated at the reactive site. When the molecule hasbeen completely synthesized, the solid support-linker-molecule constructis subjected to cleavage conditions which releases the molecule from thesolid support. The labile linkers which have been developed for use inthis context (or which may be used in this context) may also be readilyused as the linker reactant in the present invention.

Lloyd-Williams, P., et al., "Convergent Solid-Phase Peptide Synthesis",Tetrahedron Report No. 347, 49(48):11065-11133 (1993) provides anextensive discussion of linkers which are labile to actinic radiation(i.e., photolysis), as well as acid, base and other cleavage conditions.Additional sources of information about labile linkers are well known inthe art.

As described above, different linker designs will confer cleavability("lability") under different specific physical or chemical conditions.Examples of conditions which serve to cleave various designs of linkerinclude acid, base, oxidation, reduction, fluoride, thiol exchange,photolysis, and enzymatic conditions.

Examples of cleavable linkers that satisfy the general criteria forlinkers listed above will be well known to those in the art and includethose found in the catalog available from Pierce (Rockford, Ill.).Examples include:

ethylene glycobis(succinimidylsuccinate) (EGS), an amine reactivecross-linking reagent which is cleavable by hydroxylamine (1 M at 37° C.for 3-6 hours);

disuccinimidyl tartarate (DST) and sulfo-DST, which are amine reactivecross-linking reagents, cleavable by 0.015 M sodium periodate;

bis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone (BSOCOES) andsulfo-BSOCOES, which are amine reactive cross-linking reagents,cleavable by base (pH 11.6);

1,4-di-[3'-(2'-pyridyldithio(propionamido)]butane (DPDPB), apyridyldithiol crosslinker which is cleavable by thiol exchange orreduction;

N-[4-(p-azidosalicylamido)-butyl]-3'-(2'-pyridydithio)propionamide(APDP), a pyridyldithiol crosslinker which is cleavable by thiolexchange or reduction;

bis-[beta-4-(azidosalicylamido)ethyl]-disulfide, a photoreactivecrosslinker which is cleavable by thiol exchange or reduction;

N-succinimidyl-(4-azidophenyl)-1,3'dithiopropionate (SADP), aphotoreactive crosslinker which is cleavable by thiol exchange orreduction;

sulfosuccinimidyl-2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionate(SAED), a photoreactive crosslinker which is cleavable by thiol exchangeor reduction;

sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3'dithiopropionate(SAND), a photoreactive crosslinker which is cleavable by thiol exchangeor reduction.

Other examples of cleavable linkers and the cleavage conditions that canbe used to release tags are as follows. A silyl linking group can becleaved by fluoride or under acidic conditions. A 3-, 4-, 5-, or6-substituted-2-nitrobenzyloxy or 2-, 3-, 5-, or6-substituted-4-nitrobenzyloxy linking group can be cleaved by a photonsource (photolysis). A 3-, 4-, 5-, or 6-substituted-2-alkoxyphenoxy or2-, 3-, 5-, or 6-substituted-4-alkoxyphenoxy linking group can becleaved by Ce(NH₄)₂ (NO₃)₆ (oxidation). A NCO₂ (urethane) linker can becleaved by hydroxide (base), acid, or LiAlH₄ (reduction). A 3-pentenyl,2-butenyl, or 1-butenyl linking group can be cleaved by O₃, O_(S) O₄/IO₄ ⁻, or KMnO₄ (oxidation). A 2-[3-, 4-, or 5-substituted-furyl]oxylinking group can be cleaved by O₂, Br₂, MeOH, or acid.

Conditions for the cleavage of other labile linking groups include:t-alkyloxy linking groups can be cleaved by acid; methyl(dialkyl)methoxyor 4-substituted-2-alkyl-1,3-dioxlane-2-yl linking groups can be cleavedby H₃ O; 2-silylethoxy linking groups can be cleaved by fluoride oracid; 2-(X)-ethoxy (where X=keto, ester amide, cyano, NO₂, sulfide,sulfoxide, sulfone) linking groups can be cleaved under alkalineconditions; 2-, 3-, 4-, 5-, or 6-substituted-benzyloxy linking groupscan be cleaved by acid or under reductive conditions; 2-butenyloxylinking groups can be cleaved by (Ph₃ P)₃ RhCl(H), 3-, 4-, 5-, or6-substituted-2-bromophenoxy linking groups can be cleaved by Li, Mg, orBuLi; methylthiomethoxy linking groups can be cleaved by Hg²⁺ ;2-(X)-ethyloxy (where X=a halogen) linking groups can be cleaved by Znor Mg; 2-hydroxyethyloxy linking groups can be cleaved by oxidation(e.g., with Pb(OAc)₄).

Preferred linkers are those that are cleaved by acid or photolysis.Several of the acid-labile linkers that have been developed for solidphase peptide synthesis are useful for linking tags to MOIs. Some ofthese linkers are described in a recent review by Lloyd-Williams et al.(Tetrahedron 49:11065-11133, 1993). One useful type of linker is basedupon p-alkoxybenzyl alcohols, of which two, 4-ydroxymethylphenoxyaceticacid and 4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid, arecommercially available from Advanced ChemTech (Louisville, Ky.). Bothlinkers can be attached to a tag via an ester linkage to thebenzylalcohol, and to an amine-containing MOI via an amide linkage tothe carboxylic acid. Tags linked by these molecules are released fromthe MOI with varying concentrations of trifluoroacetic acid. Thecleavage of these linkers results in the liberation of a carboxylic acidon the tag. Acid cleavage of tags attached through related linkers, suchas 2,4-dimethoxy-4'-(carboxymethyloxy)-benzhydrylamine (available fromAdvanced ChemTech in FMOC-protected form), results in liberation of acarboxylic amide on the released tag.

The photolabile linkers useful for this application have also been forthe most part developed for solid phase peptide synthesis (seeLloyd-Williams review). These linkers are usually based on2-nitrobenzylesters or 2-nitrobenzylamides. Two examples of photolabilelinkers that have recently been reported in the literature are4-(4-(1-Fmoc-amino)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid (Holmesand Jones, J. Org. Chem. 60:2318-2319, 1995) and3-(Fmoc-amino)-3-(2-nitrophenyl)propionic acid (Brown et al., MolecularDiversity 1:4-12, 1995). Both linkers can be attached via the carboxylicacid to an amine on the MOI. The attachment of the tag to the linker ismade by forming an amide between a carboxylic acid on the tag and theamine on the linker. Cleavage of photolabile linkers is usuallyperformed with UV light of 350 nm wavelength at intensities and timesknown to those in the art. Cleavage of the linkers results in liberationof a primary amide on the tag. Examples of photocleavable linkersinclude nitrophenyl glycine esters, exo- and endo-2-benzonorborneylchlorides and methane sulfonates, and 3-amino-3(2-nitrophenyl) propionicacid. Examples of enzymatic cleavage include esterases which will cleaveester bonds, nucleases which will cleave phosphodiester bonds, proteaseswhich cleave peptide bonds, etc.

A preferred linker component has an ortho-nitrobenzyl structure as shownbelow: ##STR19## wherein one carbon atom at positions a, b, c, d or e issubstituted with -L³ -X, and L¹ (which is preferably a direct bond) ispresent to the left of N(R¹) in the above structure. Such a linkercomponent is susceptible to selective photo-induced cleavage of the bondbetween the carbon labeled "a" and N(R¹). The identity of R¹ is nottypically critical to the cleavage reaction, however R¹ is preferablyselected from hydrogen and hydrocarbyl. The present invention providesthat in the above structure, --N(R¹)-- could be replaced with --O--.Also in the above structure, one or more of positions b, c, d or e mayoptionally be substituted with alkyl, alkoxy, fluoride, chloride,hydroxyl, carboxylate or amide, where these substituents areindependently selected at each occurrence.

A further preferred linker component with a chemical handle L_(h) hasthe following structure: ##STR20## wherein one or more of positions b,c, d or e is substituted with hydrogen, alkyl, alkoxy, fluoride,chloride, hydroxyl, carboxylate or amide, R¹ is hydrogen or hydrocarbyl,and R² is --OH or a group that either protects or activates a carboxylicacid for coupling with another moiety. Fluorocarbon andhydrofluorocarbon groups are preferred groups that activate a carboxylicacid toward coupling with another moiety.

3. Molecule of Interest (MOI)

Examples of MOIs include nucleic acids or nucleic acid analogues (e.g.,PNA), fragments of nucleic acids (i.e., nucleic acid fragments),synthetic nucleic acids or fragments, oligonucleotides (e.g., DNA orRNA), proteins, peptides, antibodies or antibody fragments, receptors,receptor ligands, members of a ligand pair, cytokines, hormones,oligosaccharides, synthetic organic molecules, drugs, and combinationsthereof.

Preferred MOIs include nucleic acid fragments. Preferred nucleic acidfragments are primer sequences that are complementary to sequencespresent in vectors, where the vectors are used for base sequencing.Preferably a nucleic acid fragment is attached directly or indirectly toa tag at other than the 3' end of the fragment; and most preferably atthe 5' end of the fragment. Nucleic acid fragments may be purchased orprepared based upon genetic databases (e.g., Dib et al., Nature380:152-154, 1996 and CEPH Genotype Database, http://www.cephb.fr) andcommercial vendors (e.g., Promega, Madison, Wis.).

As used herein, MOI includes derivatives of an MOI that containfunctionality useful in joining the MOI to a T-L-L_(h) compound. Forexample, a nucleic acid fragment that has a phosphodiester at the 5'end, where the phosphodiester is also bonded to an alkyleneamine, is anMOI. Such an MOI is described in, e.g., U.S. Pat. No. 4,762,779 which isincorporated herein by reference. A nucleic acid fragment with aninternal modification is also an MOI. An exemplary internal modificationof a nucleic acid fragment is where the base (e.g., adenine, guanine,cytosine, thymidine, uracil) has been modified to add a reactivefunctional group. Such internally modified nucleic acid fragments arecommercially available from, e.g., Glen Research, Herndon, Va. Anotherexemplary internal modification of a nucleic acid fragment is where anabasic phosphoramidate is used to synthesize a modified phosphodiesterwhich is interposed between a sugar and phosphate group of a nucleicacid fragment. The abasic phosphoramidate contains a reactive groupwhich allows a nucleic acid fragment that contains thisphosphoramidate-derived moiety to be joined to another moiety, e.g., aT-L-L_(h) compound. Such abasic phosphoramidates are commerciallyavailable from, e.g., Clonetech Laboratories, Inc., Palo Alto, Calif.

4. Chemical Handles (L_(h))

A chemical handle is a stable yet reactive atomic arrangement present aspart of a first molecule, where the handle can undergo chemical reactionwith a complementary chemical handle present as part of a secondmolecule, so as to form a covalent bond between the two molecules. Forexample, the chemical handle may be a hydroxyl group, and thecomplementary chemical handle may be a carboxylic acid group (or anactivated derivative thereof, e.g., a hydrofluroaryl ester), whereuponreaction between these two handles forms a covalent bond (specifically,an ester group) that joins the two molecules together.

Chemical handles may be used in a large number of covalent bond-formingreactions that are suitable for attaching tags to linkers, and linkersto MOIs. Such reactions include alkylation (e.g., to form ethers,thioethers), acylation (e.g., to form esters, amides, carbamates, ureas,thioureas), phosphorylation (e.g., to form phosphates, phosphonates,phosphoramides, phosphonamides), sulfonylation (e.g., to formsulfonates, sulfonamides), condensation (e.g., to form imines, oximes,hydrazones), silylation, disulfide formation, and generation of reactiveintermediates, such as nitrenes or carbenes, by photolysis. In general,handles and bond-forming reactions which are suitable for attaching tagsto linkers are also suitable for attaching linkers to MOIs, andvice-versa. In some cases, the MOI may undergo prior modification orderivitization to provide the handle needed for attaching the linker.

One type of bond especially useful for attaching linkers to MOIs is thedisulfide bond. Its formation requires the presence of a thiol group("handle") on the linker, and another thiol group on the MOI. Mildoxidizing conditions then suffice to bond the two thiols together as adisulfide. Disulfide formation can also be induced by using an excess ofan appropriate disulfide exchange reagent, e.g., pyridyl disulfides.Because disulfide formation is readily reversible, the disulfide mayalso be used as the cleavable bond for liberating the tag, if desired.This is typically accomplished under similarly mild conditions, using anexcess of an appropriate thiol exchange reagent, e.g., dithiothreitol.

Of particular interest for linking tags (or tags with linkers) tooligonucleotides is the formation of amide bonds. Primary aliphaticamine handles can be readily introduced onto synthetic oligonucleotideswith phosphoramidites such as6-monomethoxytritylhexylcyanoethyl-N,N-diisopropyl phosphoramidite(available from Glenn Research, Sterling, Va.). The amines found onnatural nucleotides such as adenosine and guanosine are virtuallyunreactive when compared to the introduced primary amine. Thisdifference in reactivity forms the basis of the ability to selectivelyform amides and related bonding groups (e.g., ureas, thioureas,sulfonamides) with the introduced primary amine, and not the nucleotideamines.

As listed in the Molecular Probes catalog (Eugene, Oreg.), a partialenumeration of amine-reactive functional groups includes activatedcarboxylic esters, isocyanates, isothiocyanates, sulfonyl halides, anddichlorotriazenes. Active esters are excellent reagents for aminemodification since the amide products formed are very stable. Also,these reagents have good reactivity with aliphatic amines and lowreactivity with the nucleotide amines of oligonucleotides. Examples ofactive esters include N-hydroxysuccinimide esters, pentafluorophenylesters, tetrafluorophenyl esters, and p-nitrophenyl esters. Activeesters are useful because they can be made from virtually any moleculethat contains a carboxylic acid. Methods to make active esters arelisted in Bodansky (Principles of Peptide Chemistry (2d ed.), SpringerVerlag, London, 1993).

5. Linker Attachment

Typically, a single type of linker is used to connect a particular setor family of tags to a particular set or family of MOIs. In a preferredembodiment of the invention, a single, uniform procedure may be followedto create all the various T-L-MOI structures. This is especiallyadvantageous when the set of T-L-MOI structures is large, because itallows the set to be prepared using the methods of combinatorialchemistry or other parallel processing technology. In a similar manner,the use of a single type of linker allows a single, uniform procedure tobe employed for cleaving all the various T-L-MOI structures. Again, thisis advantageous for a large set of T-L-MOI structures, because the setmay be processed in a parallel, repetitive, and/or automated manner.

There are, however, other embodiment of the present invention, whereintwo or more types of linker are used to connect different subsets oftags to corresponding subsets of MOIs. In this case, selective cleavageconditions may be used to cleave each of the linkers independently,without cleaving the linkers present on other subsets of MOIs.

A large number of covalent bond-forming reactions are suitable forattaching tags to linkers, and linkers to MOIs. Such reactions includealkylation (e.g., to form ethers, thioethers), acylation (e.g., to formesters, amides, carbamates, ureas, thioureas), phosphorylation (e.g., toform phosphates, phosphonates, phosphoramides, phosphonamides),sulfonylation (e.g., to form sulfonates, sulfonamides), condensation(e.g., to form imines, oximes, hydrazones), silylation, disulfideformation, and generation of reactive intermediates, such as nitrenes orcarbenes, by photolysis. In general, handles and bond-forming reactionswhich are suitable for attaching tags to linkers are also suitable forattaching linkers to MOIs, and vice-versa. In some cases, the MOI mayundergo prior modification or derivitization to provide the handleneeded for attaching the linker.

One type of bond especially useful for attaching linkers to MOIs is thedisulfide bond. Its formation requires the presence of a thiol group("handle") on the linker, and another thiol group on the MOI. Mildoxidizing conditions then suffice to bond the two thiols together as adisulfide. Disulfide formation can also be induced by using an excess ofan appropriate disulfide exchange reagent, e.g., pyridyl disulfides.Because disulfide formation is readily reversible, the disulfide mayalso be used as the cleavable bond for liberating the tag, if desired.This is typically accomplished under similarly mild conditions, using anexcess of an appropriate thiol exchange reagent, e.g., dithiothreitol.

Of particular interest for linking tags to oligonucleotides is theformation of amide bonds. Primary aliphatic amine handles can be readilyintroduced onto synthetic oligonucleotides with phosphoramidites such as6-monomethoxytritylhexylcyanoethyl-N,N-diisopropyl phosphoramidite(available from Glenn Research, Sterling, Va.). The amines found onnatural nucleotides such as adenosine and guanosine are virtuallyunreactive when compared to the introduced primary amine. Thisdifference in reactivity forms the basis of the ability to selectivelyform amides and related bonding groups (e.g., ureas, thioureas,sulfonamides) with the introduced primary amine, and not the nucleotideamines.

As listed in the Molecular Probes catalog (Eugene, Oreg.), a partialenumeration of amine-reactive functional groups includes activatedcarboxylic esters, isocyanates, isothiocyanates, sulfonyl halides, anddichlorotriazenes. Active esters are excellent reagents for aminemodification since the amide products formed are very stable. Also,these reagents have good reactivity with aliphatic amines and lowreactivity with the nucleotide amines of oligonucleotides. Examples ofactive esters include N-hydroxysuccinimide esters, pentafluorophenylesters, tetrafluorophenyl esters, and p-nitrophenyl esters. Activeesters are useful because they can be made from virtually any moleculethat contains a carboxylic acid. Methods to make active esters arelisted in Bodansky (Principles of Peptide Chemistry (2d ed.), SpringerVerlag, London, 1993).

Numerous commercial cross-linking reagents exist which can serve aslinkers (e.g., see Pierce Cross-linkers, Pierce Chemical Co., Rockford,Ill.). Among these are homobifunctional amine-reactive cross-linkingreagents which are exemplified by homobifunctional imidoesters andN-hydroxysuccinimidyl (NHS) esters. There also exist heterobifunctionalcross-linking reagents possess two or more different reactive groupsthat allows for sequential reactions. Imidoesters react rapidly withamines at alkaline pH. NHS-esters give stable products when reacted withprimary or secondary amines. Maleimides, alkyl and aryl halides,alpha-haloacyls and pyridyl disulfides are thiol reactive. Malcimidesare specific for thiol (sulfhydryl) groups in the pH range of 6.5 to7.5, and at alkaline pH can become amine reactive. The thioether linkageis stable under physiological conditions. Alpha-haloacetyl cross-linkingreagents contain the iodoacetyl group and are reactive towardssulfhiydryls. Imidazoles can react with the iodoacetyl moiety, but thereaction is very slow. Pyridyl disulfides react with thiol groups toform a disulfide bond. Carbodiimides couple carboxyls to primary aminesof hydrazides which give rises to the formation of an acyl-hydrazinebond. The arylazides are photoaffinity reagents which are chemicallyinert until exposed to UV or visible light. When such compounds arephotolyzed at 250-460 nm, a reactive aryl nitrene is formed. Thereactive aryl nitrene is relatively non-specific. Glyoxals are reactivetowards guanidinyl portion of arginine.

In one typical embodiment of the present invention, a tag is firstbonded to a linker, then the combination of tag and linker is bonded toa MOI, to create the structure T-L-MOI. Alternatively, the samestructure is formed by first bonding a linker to a MOI, and then bondingthe combination of linker and MOI to a tag. An example is where the MOIis a DNA primer or oligonucleotide. In that case, the tag is typicallyfirst bonded to a linker, then the T-L is bonded to a DNA primer oroligonucleotide, which is then used, for example, in a sequencingreaction.

One useful form in which a tag could be reversibly attached to an MOI(e.g., an oligonucleotide or DNA sequencing primer) is through achemically labile linker. One preferred design for the linker allows thelinker to be cleaved when exposed to a volatile organic acid, forexample, trifluoroacetic acid (TFA). TFA in particular is compatiblewith most methods of MS ionization, including electrospray.

The invention compositions for mutation analysis. A composition usefulfor mutation analysis comprises a pair of compounds of the formula:

    T.sup.ms -L-MOI

wherein T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine. Inthe formula, L is an organic group which allows a T^(ms) -containingmoiety to be cleaved from the remainder of the compound, wherein theT^(ms) -containing moiety comprises a functional group which supports asingle ionized charge state when the compound is subjected to massspectrometry and is selected from tertiary amine, quaternary amine andorganic acid. In the formula, MOI is a nucleic acid fragment wherein Lis conjugated to MOI at other than the 3' end of the MOI. Thecomposition comprises pairs of compounds where the members of a pairhave non-identical T^(ms) groups, and have identical sequences except atone base position where the bases are non-identical. In anotherembodiment of the inventive composition, the member of the pairs ofcompounds have non-identical T^(ms) groups, and have identical sequencesexcept at one base position where the bases are non-identical. Thesecompositions are then added to a support-bound nucleic acid sequence,which is identical to the sequence of one of the members of each pair.Thus, the invention provides for a composition comprising a plurality ofcompound pairs as described above, and further comprising an equalplurality of nucleic acids immobilized on a solid support, wherein eachmember of the plurality of nucleic acids has a base sequence that isexactly complementary to one member of each of the pairs.

The invention also provides a kit for mutation analysis comprising aplurality of containers. Each container comprises a pair of compounds ofthe formula:

    T.sup.ms -L-MOI

wherein T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine. Inthe formula, L is an organic group which allows a T^(ms) -containingmoiety to be cleaved from the remainder of the compound, wherein theT^(ms) -containing moiety comprises a functional group which supports asingle ionized charge state when the compound is subjected to massspectrometry and is selected from tertiary amine, quaternary amine andorganic acid. In the formula, MOI is a nucleic acid fragment wherein Lis conjugated to MOI at other than the 3' end of the MOI. In the kit,the compounds of each pair have non-identical T^(ms) groups, and haveidentical sequences except at one or two base position where the basesare non-identical. In a preferred kit, the plurality is at least 3, andmore preferably is at least 5.

Assays

As noted above, the present invention a wide variety of assays whereinthe tags and detection methodology provided herein can be utilized inorder to greatly increase the sensitivity and throughput of the assay.Within one aspect, such methods can be utilized to detect the binding ofa first member to a second member of a ligand pair, comprising the stepsof (a) combining a set of first tagged members with a biological samplewhich may contain one or more second members, under conditions, and fora time sufficient to permit binding of a first member to a secondmember, wherein said tag is correlative with a particular first memberand detectable by non-fluorescent spectrometry, or potentiometry, (b)separating bound first and second members from unbound members, (c)cleaving the tag from the tagged first member, and (d) detecting the tagby non-fluorescent spectrometry, or potentiometry, and therefromdetecting the binding of the first member to the second member.

A wide variety of first and second member pairs may be utilized withinthe context of the present invention, including for example, nucleicacid molecules (e.g., DNA, RNA, nucleic acid analogues such as PNA, orany combination of these), proteins or polypeptides (e.g., an antibodiesor antibody fragments (e.g., monoclonal antibodies, polyclonalantibodies, or binding partners such as a CDR), oligosaccharides,hormones, organic molecules and other substrates (e.g., xenobiotics suchas glucuronidase--drug molecule), or any other ligand of a ligand pair.Within various embodiments of the invention, the first and secondmembers may be the same type of molecule or of different types. Forexample, representative first member second member ligand pairs include:nucleic acid molecule/nucleic acid molecule; antibody/nucleic acidmolecule; antibody/hormone; antibody/xenobiotic; and antibody/protein.

In order to further an understanding of assays which can be accomplishedgiven the disclosure provided herein, a brief discussion is providedbelow of certain particularly preferred assays.

6. Nucleic Acid Assays

a. Introduction

As noted above, the present invention also provides a wide variety ofmethods wherein the above-described cleavable tags and/or linkers may beutilized in place of traditional labels (e.g., radioactive, fluorescent,or enzymatic), in order enhance the specificity, sensitivity, or numberof samples that may be simultaneously analyzed, within a given method.Representative examples of such methods which may be enhanced include,for example, standard nucleic acid hybridization reactions (see Sambrooket al., supra), diagnostic reactions such as Cycling Probe Technology(CPT) (see U.S. Pat. Nos. 4,876,187 and 5,011,769) orOligonucleotide-Ligation Assay (OLA) (Burket et al., Science 196:180,1987). These as well as other techniques are discussed in more detailbelow.

b. Hybridization Techniques

The successful cloning and sequencing of a gene allows investigation ofits structure and expression by making it possible to detect the gene orits mRNA in a large pool of unrelated DNA or RNA molecules. The amountof mRNA encoding a specific protein in a tissue is an importantparameter for the activity of a gene and may be significantly related tothe activity of function systems. Its regulation is dependent upon theinteraction between sequences within the gene (cis-acting elements) andsequence-specific DNA binding proteins (trans-acting factors), which areactivated tissue-specifically or by hormones and second messengersystems.

Several techniques are available for analysis of a particular gene, itsregulatory sequences, its specific mRNA and the regulation of itsexpression; these include Southern or Northern blot analysis,ribonuclease (RNase) protection assay and in situ hybridization.

Variations in the nucleotide composition of a certain gene may be ofgreat pathophysiological relevance. When localized in the non-codingregions (5', 3'-flanking regions and intron), they can affect theregulation of gene expression, causing abnormal activation orinhibition. When localized in the coding regions of the gene (exons),they may result in alteration of the protein function or dysfunctionalproteins.

Thus, a certain sequence within a gene can correlate to a specificdisease and can be useful as a marker of the disease. One primary goalof research in the medical field is, therefore, to detect those geneticvariations as diagnostic tools, and to gain important information forthe understanding of pathophysiological phenomena.

The basic method for the analysis of a population regarding thevariations within a certain gene is DNA analysis using the Southern blottechnique. Briefly, prepared genomic DNA is digested with a restrictionenzyme (RE), resulting in a large number of DNA fragments of differentlengths, determined by the presence of the specific recognition site ofthe RE on the genome. Alleles of a certain gene with mutations insidethis restriction site will be cleaved into fragments of different numberand length. This is called restriction fragment length polymorphism(RFLP) and can be an important diagnostic marker with many applications.

The fragment to be analyzed has to bc separated from the pool of DNAfragments and distinguished from other DNA species using a specificprobe. Thus, DNA is subjected to electrophoretic fractionation using anagarose gel, followed by transfer and fixation to a nylon ornitrocellulose membrane. The fixed, single-stranded DNA is hybridized toa tagged DNA which is complementary to the DNA to be detected. Afterremoving non-specific hybridizations, the DNA fragment of interest canbe visualized by MALD1-MS as described in more detail below.

The presence and quantification of a specific gene transcript and itsregulation by physiological parameters can be analysed by means ofNorthern blot analysis and RNase protection assay.

The principle basis of these methods is hybridization of a pool of totalcellular RNA to a specific probe. In the Northern blot technique, totalRNA of a tissue is electrophoretically fractionated using an agarosegel, transferred and immobilized to a labeled antisense RNA (cRNA),complementary to the RNA to be detected. This cRNA probe is then taggedas described herein. By applying stringent washing conditions,non-specifically bound molecules are eliminated. Specifically boundmolecules, which can subsequently be detected by MALD1-MS. In addition,specificity can be controlled by comparing the size of the detected mRNAwith the predicted length of the mRNA of interest.

More rapid, but less specific, is the dot blot method, which isperformed as the Northern blot technique except that the RNA is directlydotted onto the membrane without preceding fractionation. The RNA isimmobilized nonspecifically in the dot blot.

The most specific method for detection of an mRNA species is the RNaseprotection assay. Briefly, total RNA from a tissue or cell culture ishybridized to a tagged specific cRNA of complete homology. Specificityis accomplished by subsequent RNase digestion. Non-hybridized,single-stranded RNA and non-specifically hybridized fragments with evensmall mismatches will be recognized and cleaved, while double-strandedRNA of complete homology is not accessible to the enzyme and will beprotected. After removing RNase by proteinase K digestion and phenolextraction, the specific protected fragment can be separated fromdegradation products, usually on a denaturing polyacrylamide gel, andthe predicted size can be checked by HPLC. All the assays describedabove can be quantified by non-fluorescent spectrometry orpotentiometry.

The precise location of a given mRNA in a specific population of cellswithin a tissue can be determined by in situ hybridization. This methodis analogous with the immunocytochemical technique and can in fact beused simultaneously with immunocytochemistry on the same section todiscover, for example, whether a certain protein is really synthesizedlocally or actually taken up from other sources. Apart from thepossibility of identifying the cell type expressing a specific mRNA, insitu hybridization can be even more sensitive than analysis of a totaltissue RNA preparation using the techniques described above. This is thecase when the mRNA is expressed in high concentrations in a verydiscrete region or cell type within the tissue and would be diluted byhomogenization of the whole tissue. The analysis of gene expression byin situ hybridization is therefore of particular importance forheterogeneous tissues like the brain. For in situ hybridization, thetissues have to be frozen or perfusion-fixed and sectioned according tohistochemical protocol. The hybridization protocol for tissue sectionsand the labeled probes used are similar to the other hybridizationmethods described above. A semiquantitative analysis is possible.

c. cDNAs as Representative Populations of mRNAs and use as Probes

Most mRNAs are transcribed from single copy sequences. Another propertyof cDNAs is that they represent a longer region of the genome because ofthe introns present in the chromosomal version of most genes. Therepresentation varies from one gene to another but can be verysignificant as many genes cover more than 100 kb in genomic DNA,represented in a single cDNA. One possible use of molecularhybridization is the use of probes from one species to find clones madefrom another species. Sequence divergence between the mRNAs of mouse andman permits specific cross-reassociation of long sequences, but exceptfor the most highly conserved regions, prevents cross-hybridization ofPCR primers.

Differential screening in complex biological samples such as developingnervous system using cDNA probes prepared from single cells is nowpossible due to the development of PCR-based and cRNA-basedamplification techniques. Several groups reported previously thegeneration of cDNA libraries from small amounts of poly (A)+ RNA (1 ngor less) prepared from 10-50 cells (Belyav et al., Nuc. Acids Res.17:2919, 1989). Although the libraries were sufficiently representativeof mRNA complexity, the average cDNA insert size of these libraries wasquite small (<2 kb).

More recently, methodologies have been combined to generate bothPCR-based (Lambolez et al., Neuron 9:247, 1992) and cRNA-based (VanGelder et al., Proc. Natl. Acad. Sci. USA 87:1663, 1990) probes fromsingle cells. After electrical recordings, the cytoplasmic contents of asingle cell were aspirated with patch-clamp microelectrodes for in situcDNA synthesis and amplification. PCR was used to amplify cDNA ofselective glutamate receptor mRNAs from single Purkinje cells and GFAPmRNA from single glia in organotypic cerebellar culture (Lambolez etal., Neuron 9:247, 1992). In the case of cRNA amplification,transcription promoter sequences were designed into primers for cDNAsynthesis and complex antisense cRNAs were generated by in vitrotranscription with bacteriophage RNA polymerases.

Thus, within one embodiment of the invention, tagged cRNAs can beutilized as tagged probes to screen cDNA libraries randomly or in"expression profiling" experiments to screen Southern blots containingcDNA fragments of interest (receptors, growth factors, ion channelsetc.). It appears that the lack of linearity of amplification, oftenencountered with PCR-based approaches, is minimized with cRNA-basedmethods.

d. Oligonucleotide-Ligation Assay

Oligonucleotide-ligation assay is an extension of PCR-based screeningthat uses an ELISA-based assay (OLA, Nickerson et al., Proc. Natl. Acad.Sci. USA 87:8923, 1990) to detect the PCR products that contain thetarget sequence. Thus, both gel electrophoresis and colony hybridizationare eliminated. Briefly, the OLA employs two adjacent oligonucleotides:a "reporter" probe (tagged at the 5' end) and a5'-phosphorylated/3'-biotinylated "anchor" probe. The twooligonucleotides, which are complementary to sequences internal to thePCR primers, are annealed to target DNA and, if there is perfectcomplementarity, the two probes are ligated by T4 DNA ligase. Capture ofthe biotinylated anchor probe on immobilized streptavidin and analysisfor the covalently linked reporter probe test for the presence orabsence of the target sequences among the PCR products.

e. Application of Hybridization Techniques

i. Forensics

The identification of individuals at the level of DNA sequence variationoffers a number of practical advantages over such conventional criteriaas fingerprints, blood type, or physical characteristics. In contrast tomost phenotypic markers, DNA analysis readily permits the deduction ofrelatedness between individuals such as is required in paternitytesting. Genetic analysis has proven highly useful in bone marrowtransplantation, where it is necessary to distinguish between closelyrelated donor and recipient cells. Two types of probes are now in usefor DNA fingerprinting by DNA blots. Polymorphic minisatellite DNAprobes identify multiple DNA sequences, each present in variable formsin different individuals, thus generating patterns that are complex andhighly variable between individuals. VNTR probes identify singlesequences in the genome, but these sequences may be present in up to 30different forms in the human population as distinguished by the size ofthe identified fragments. The probability that unrelated individualswill have identical hybridization patterns for multiple VNTR orminisatellite probes is very low. Much less tissue than that requiredfor DNA blots, even single hairs, provides sufficient DNA for aPCR-based analysis of genetic markers. Also, partially degraded tissuemay be used for analysis since only small DNA fragments are needed.Forensic DNA analyses will eventually be carried out with polymorphicDNA sequences that can be studied by simple automatable assays such asOLA. For example, the analysis of 22 separate gene sequences, each onepresent in two different forms in the population, could generate 1010different outcomes, permitting the unique identification of humanindividuals.

ii. Tumor diagnostics

The detection of viral or cellular oncogenes is another important fieldof application of nucleic acid diagnostics. Viral oncogenes(v-oncogenes) are transmitted by retroviruses while their cellularcounterparts (c-oncogenes) are already present in normal cells. Thecellular oncogenes can, however, be activated by specific modificationssuch s point mutations (as in the c-K-ras oncogene in bladder carcinomaand in colorectal tumors), promoter induction, gene amplification (as inthe N-myc oncogene in the case of neuroblastoma) or the rearrangement ofchromosomes (as in the translocation of the c-abl oncogene fromchromosome 9 to chromosome 22 in the case of chronic myeloid leukemia).Each of the activation processes leads, in conjunction with additionaldegenerative processes, to an increased and uncontrolled cell growth.The so-called "recessive oncogenes" which must be inactivated for theformation of a tumor (as in the retinoblastoma (Rb gene and theosteosarcoma can also be detected with the help of DNA probes. Usingprobes against immunoglobulin genes and against T-cell receptor genes,the detection of B-cell lymphomas and lymphoblastic leukemia ispossible.

iii. Transplantation analyses

The rejection reaction of transplanted tissue is decisively controlledby a specific class of histocompatibility antigens (HLA). They areexpressed on the surface of antigen-presenting blood cells, e.g.,macrophages. The complex between the HLA and the foreign antigen isrecognized by T-helper cells through corresponding T-cell receptors onthe cell surface. The interaction between HLA, antigen and T-cellreceptor triggers a complex defense reaction which leads to acascade-like immune response on the body.

The recognition of different foreign antigens is mediated by variable,antigen-specific regions of the T-cell receptor--analogous to theantibody reaction. In a graft rejection, the T-cells expressing aspecific T-cell receptor which fits to the foreign antigen, couldtherefore be eliminated from the T-cell pool. Such analyses are possibleby the identification of antigen-specific variable DNA sequences whichare amplified by PCR and hence selectively increased. The specificamplification reaction permits the single cell-specific identificationof a specific T-cell receptor.

Similar analyses are presently performed for the identification ofauto-immune disease like juvenile diabetes, arteriosclerosis, multiplesclerosis, rheumatoid arthritis, or encephalomyelitis.

iv. Genome Diagnostics

Four percent of all newborns are born with genetic defects; of the 3,500hereditary diseases described which are caused by the modification ofonly a single gene, the primary molecular defects are only known forabout 400 of them.

Hereditary diseases have long since been diagnosed by phenotypicanalyses (anamneses, e.g., deficiency of blood: thalassemias),chromosome analyses (karyotype, e.g., mongolism: trisomy 21) or geneproduct analyses (modified proteins, e.g., phenylketonuria: deficiencyof the phenylalanine hydroxylase enzyme resulting in enhanced levels ofphenylpyruvic acid). The additional use of nucleic acid detectionmethods considerably increases the range of genome diagnostics.

In the case of certain genetic diseases, the modification of just one ofthe two alleles is sufficient for disease (dominantly transmittedmonogenic defects); in many cases, both alleles must be modified(recessively transmitted monogenic defects). In a third type of geneticdefect, the outbreak of the disease is not only determined by the genemodification but also by factors such as eating habits (in the case ofdiabetes or arteriosclerosis) or the lifestyle (in the case of cancer).Very frequently, these diseases occur in advanced age. Diseases such asschizophrenia, manic depression or epilepsy should also be mentioned inthis context; it is under investigation if the outbreak of the diseasein these cases is dependent upon environmental factors as well as on themodification of several genes in different chromosome locations.

Using direct and indirect DNA analysis, the diagnosis of a series ofgenetic diseases has become possible: sickle-cell anemia, thalassemias,a1-antitrypsin deficiency, Lesch-Nyhan syndrome, cysticfibrosis/mucoviscidosis, Duchenne/Becker muscular dystrophy, Alzheimer'sdisease, X-chromosome-dependent mental deficiency, Huntington's chorea

v. Infectious Disease

The application of recombinant DNA methods for diagnosis of infectiousdiseases has been most extensively explored for viral infections wherecurrent methods are cumbersome and results are delayed. In situhybridization of tissues or cultured cells has made diagnosis of acuteand chronic herpes infection possible. Fresh and fomalin-fixed tissueshave been reported to be suitable for detection of papillomavirus ininvasive cervical carcinoma and in the detection of HIV, while culturedcells have been used for the detection of cytomegalovirus andEpstein-Barr virus. The application of recombinant DNA methods to thediagnosis of microbial diseases has the potential to replace currentmicrobial growth methods if cost-effectiveness, speed, and precisionrequirements can be met. Clinical situations where recombinant DNAprocedures have begun to be applied include the identification ofpenicillin-resistant Neisseria gonorrhoeae by the presence of atransposon, the fastidiously growing chlamydia, microbes in foods; andsimple means of following the spread of an infection through apopulation. The worldwide epidemiological challenge of diseasesinvolving such parasites as leishmania and plasmodia is already beingmet by recombinant methods.

7. Protein-Based Assays

a. Introduction

As noted above, a wide variety of protein based assays may likewise beenhanced by the tags described herein (see, e.g., Antibodies: ALaboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor LaboratoryPress, 1988. Representative examples include antigen--antibody assayssuch as: countercurrent immuno-electrophoresis (CIEP), enzyme-linkedimmuno-sorbent assays (ELISA), inhibition or competition assays, andsandwich assays, simultaneous immunoassays and immunofiltration assays.A wide variety of other assays however may likewise be enhance,including for example, ligand--receptor assays and the like.

b. Immunoassays

Since the development of RIAs for insulin and thyroxin, methodsinvolving radioisotopically labeled antigens have been widely applied inthe measurement of haptenic molecules such as hormones and drugs. Themethods are based on the competition between a labeled antigen and anunlabeled antigen for a limited amount of antibody. These methods mightalso be described as "limited reagent" methods because of the limitedamount of antibody used in the assay.

Although labeled antibodies have been used in immunofluorescence methodssince 1941, they were not more widely applied in quantitative methodsuntil the introduction of radioisotope-labeled antibodies in IRMA.IRMAs, as well as other solid-phase-based double-antibody or "sandwich"assays (ELISA, IFMA, immunofluoresence staining assays), arecharacterized by an excess of antibodies over antigens; they could thusbe called "excess reagent" methods. In principle, using excess reagentsshortens the incubation time and potentially increases sensitivity. Thesolid phase facilitates separation, and the signal is directlyproportional to the amount of antigen--as opposed to the inverserelationship in competitive assays.

The use of avidin-biotin technology has become increasingly important innumerous areas of biochemistry, molecular biology, and medicine,including detection of proteins by nonradioactive immunoassays,cytochemical staining, cell separation, and isolation of nucleic acidsand detection of specific DNA/RNA sequences by hybridization. Thetechnique derives its usefulness from the extremely high affinity of theavidin-biotin interaction (association constant 1015M-1) and the abilityto biotinylate a wide range of target biomolecules such as antibodies,nucleic acids, and lipids. The first step in the isolation of a targetmolecule is its biotinylation or the biotinylation of a biomoleculewhich ultimately binds to the target molecule (e.g., an antibody orhybridization probe that forms a target complex). The biotinylatedmolecule or the target complex is then separated from other molecules ina heterogeneous mixture by using affinity media based on theavidin-biotin interactions.

Thus, within one embodiment of the invention any of the standardimmunoassays may be accomplished utilized tagged reagents, rather thanthe typical isotopically labeled reagents. Such methods result ingreatly increased sensitivity, as well as the capability of analyzingmany samples simultaneously.

8. Gene Expression Analysis

One of the inventions disclosed herein is a high through-put method formeasuring the expression of numerous genes (1-2000) in a singlemeasurement. The method also has the ability to be done in parallel withgreater than one hundred samples per process. The method is applicableto drug screening, developmental biology, molecular medicine studies andthe like. Thus, within one aspect of the invention methods are providedfor analyzing the pattern of gene expression from a selected biologicalsample, comprising the steps of (a) exposing nucleic acids from abiological sample, (b) combining the exposed nucleic acids with one ormore selected tagged nucleic acid probes, under conditions and for atime sufficient for said probes to hybridize to said nucleic acids,wherein the tag is correlative with a particular nucleic acid probe anddetectable by non-fluorescent spectrometry, or potentiometry, (c)separating hybridized probes from unhybridized probes, (d) cleaving thetag from the tagged fragment, and (e) detecting the tag bynon-fluorescent spectrometry, or potentiometry, and therefromdetermining the patter of gene expression of the biological sample.

Within a particularly preferred embodiment of the invention, assays ormethods are provided which are described as follows: RNA from a targetsource is bound to a solid support through a specific hybridization step(i.e., capture of poly(A) mRNA by a tethered oligo(dT) capture probe).The solid support is then washed and cDNA is synthesized on the solidsupport using standard methods (i.e., reverse transcriptase). The RNAstrand is then removed via hydrolysis. The result is the generation of aDNA population which is covalently immobilized to the solid supportwhich reflects the diversity, abundance, and complexity of the RNA fromwhich the cDNA was synthesized. The solid support then interrogated(hybridized) with 1 to several thousand probes which are complementaryto a gene sequence of interest. Each probe type is labelled with acleavable mass spectrometry tag or other type of cleavable tag. Afterthe interrogation step, excess or unhybridized probe is washed away, thesolid support is placed (for example) in the well of a microtiter plateand the mass spectrometry tag is cleaved from the solid support. Thesolid support is removed from the well of sample container, and thecontents of the well are measured with a mass spectrometer. Theappearance of specific mass spectrometer tags indicate the presence ofRNA in the sample and evidence that a specific gene is expressed in agiven biological sample. The method can also be quantifiable.

The compositions and methods for the rapid measurement of geneexpression using cleavable tags can be described in detail as follows.Briefly, tissue (liver, muscle, etc.), primary or transformed celllines, isolated or purified cell types or any other source of biologicalmaterial in which determining genetic expression is useful can be usedas a source of RNA. In the preferred method, the biological sourcematerial is lysed in the presence of a chaotrope in order to suppressnucleases and proteases and support stringent hybridization of targetnucleic acid to the solid support. Tissues, cells and biological sourcescan be effectively lysed in 1 to 6 molar chaotropic salts (guanidinehydrochloride, guanidine thiocyanate, sodium perchlorate, etc.). Afterthe source biological sample is lysed, the solution is mixed with asolid support to effect capture of target nucleic acid present in thelysate. In one permutation of the method, RNA is captured using atethered oligo(dT) capture probe. Solid supports can include nylonbeads, polystyrene microbeads, glass beads and glass surfaces or anyother type of solid support to which oligonucleotides can be covalentlyattached. The solid supports are preferentially coated with anamine-polymer such as polyethylene(imine), acrylamide, amine-dendrimers,etc. The amines on the polymers are used to covalently immobilizeoligonucleotides. Oligonucleotides are preferentially synthesized with a5'-amine (generally a hexylamine which is includes a six carbonspacer-arm and a distal amine). Oligonucleotides can be 15 to 50nucleotides in length. Oligonucleotides are activated withhomo-bifunctional or hetero-bifunctional cross-linking reagents such ascyanuric chloride. The activated oligonucleotides are purified fromexcess cross-linking reagent (i.e., cyanuric chloride) by exclusionchromatography. The activated oligonucleotide are then mixed with thesolid supports to effect covalent attachment. After covalent attachmentof the oligonucleotides, the unreacted amines of the solid support arecapped (i.e., with succinic anhydride) to eliminate the positive chargeof the solid support.

The solid supports can be used in parallel and are preferentiallyconfigured in a 96-well or 384-well format. The solid supports can beattached to pegs, stems, or rods in a 96-well or 384-well configuration,the solid supports either being detachable or alternatively integral tothe particular configuration. The particular configuration of the soldsupports is not of critical importance to the functioning of the assay,but rather, affects the ability of the assay to be adapted toautomation.

The solid supports are mixed with the lysate for 15 minutes to severalhours to effect capture of the target nucleic acid onto the solidsupport. In general, the "capture" of the target nucleic acid is throughcomplementary base pairing of target RNA and the capture probeimmobilized on the solid support. One permutation utilizes the 3'poly(A) stretch found on most eucaryotic messengers RNAs to hybridize toa tethered oligo(dT) on the solid support. Another permutation is toutilize a specific oligonucleotide or long probes (greater than 50bases) to capture an RNA containing a defined sequence. Anotherpossibility is to employ degenerate primers (oligonucleotides) thatwould effect the capture of numerous related sequences in the target RNApopulation. Hybridization times are guided by the sequence complexity ofthe RNA population and the type of capture probe employed. Hybridizationtemperatures are dictated by the type of chaotrope employed and thefinal concentration of chaotrope (see Van Ness and Chen, Nuc. Acids Res.for general guidelines). The lysate is preferentially agitated with thesolid support continually to effect diffusion of the target RNA. Oncethe step of capturing the target nucleic acid is accomplished, thelysate is washed from the solid support and all chaotrope orhybridization solution is removed. The solid support is preferentiallywashed with solutions containing ionic or non-ionic detergents, buffersand salts. The next step is the synthesis of DNA complementary to thecaptured RNA. In this step, the tethered capture oligonucleotide servesas the extension primer for reverse transcriptase. The reaction isgenerally performed at 25 to 37° C. and preferably agitated during thepolymerization reaction. After the cDNA is synthesized, it becomescovalently attached to the solid support since the captureoligonucleotide serves as the extension primer. The RNA is thenhydrolysed from the cDNA/RNA duplex. The step can be effected by the useof heat which denatures the duplex or the use of base (i.e., 0.1 N NaOH)to chemically hydrolyse the RNA. The key result at this step is to makethe cDNA available for subsequent hybridization with defined probes. Thesolid support or set of solid supports are then further washed to removeRNA or RNA fragments. At this point the solid support contains aapproximate representative population of cDNA molecules that representsthe RNA population in terms of sequence abundance, complexity, anddiversity.

The next step is to hybridize selected probes to the solid support toidentify the presence or absence and the relative abundance specificcDNA sequences. Probes are preferentially oligonucleotides in length of15 to 50 nucleotides. The sequence of the probes is dictated by theend-user of the assay. For example, if the end-user intended to studygene expression in an inflammatory response in a tissue, probes would beselected to be complementary to numerous cytokine mRNAs, RNAs thatencode enzymes that modulate lipids, RNAs that encode factors thatregulate cells involved in an inflammatory response, etc. Once a set ofdefined sequences are defined for study, each sequence is made into anoligonucleotide probe and each probe is assigned a specific cleavabletag. The tag(s) is then attached to the respective oligonucleotide(s).The oligonucleotide(s) are then hybridized to the cDNA on the solidsupport under appropriate hybridization conditions. After completion ofthe hybridization step, the solid support is washed to remove anyunhybridized probe. The solid support or array of supports are thenplace in solutions which effect the cleavage of the mass spectrometertags. The mass spectrometer tags are then subjected to measurement by amass spectrometer, the mass each tag present is identified, and thepresence (and abundance) or absence of an expressed mRNA is determined.

9. Detection of Micro-Organisms, Specific Gene Expression or SpecificSequences in Nucleic Acid

The use of DNA probes with cleavable tags can be used to detect thepresence or absence of micro-organisms in any type of sample orspecimen. Typically, the sample will be subjected to a lysis step usingionic detergents or choatropes, the nucleic acid is then specifically ornon-specifically immobilized on a solid support, and then probed withtagged DNA probes. Unhybridized probe is removed is a washing step, thetags are cleaved form their respective probes, and the measured.

Detectable nucleic acid can include mRNA, genomic DNA, plasmid DNA orRNA, rRNA viral DNA or RNA. To effect detection of the target nucleicacid, the target requires some type of immobilization since the assaysdescribed herein are not homogeneous. Two types of immobilization arepossible, non-specific or specific. In the former case nucleic acids areimmobilized on solid support or substrate which possesses some affinityfor nucleic acid. The nucleic acids can be purified or not purifiedprior to non-specific immobilization. Solid supports can include nylonmembranes, membranes composed of nitrocellulose, etc. The solid supportsare then probed with tagged oligonucleotides of pre-determined sequenceto identify the target nucleic acid of interest. Unhybridized probe isremoved is a washing step, the tags are cleaved form their respectiveprobes, and then measured.

Another method which results in higher specificity for the analysis of apopulation regarding the presence of a certain gene or DNA sequenceutilizes the Southern blot technique. Prepared DNA is digested with arestriction enzyme (RE), resulting in a large number of DNA fragments ofdifferent lengths, determined by the presence of the specificrecognition site of the restriction enzyme on the genome. Alleles of acertain gene with mutations inside this restriction site will be cleavedinto fragments of different number and length. The resulting restrictionfragment length polymorphism (RFLP) can be an important diagnostic of amicro-organism if the fragment can be specifically identified.

The fragment to be analyzed should be separated from the pool of DNAfragments and distinguished from other DNA species using specificprobes. Thus, DNA is subjected to electrophoretic fractionation usingsome type of gel or chromatography, followed by transfer and fixation toa nylon or nitrocellulose membrane. The fixed, single-stranded DNA ishybridized to a tagged oligonucleotide, complementary to the DNA to bedetected. After removing non-specific hybridizations, the DNA fragmentof interest is identified by cleaving the tag(s) from the hybridizedprobe. With the technology described here, over one hundred probes canbe used simultaneously.

The presence and quantification of a specific gene transcripts can beanalysed by means of Northern blot analysis and RNase protection assay.The principle basis of these methods is hybridization of the pool oftotal cellular RNA to a specific tagged probe or set of specific taggedprobes. In the Northern blot technique, total RNA of a tissue iselectrophoretically fractionated using an agarose gel, transferred andimmobilized to a solid support (nylon, nitrocellulose, etc.). The RNA ishybridized to a tagged oligonucleotide, complementary to the RNA to bedetected. After removing non-specific hybridizations, the RNA fragmentof interest is identified by cleaving the tag(s) from the hybridizedprobe. By applying stringent washing conditions, non-specifically boundmolecules are eliminated due to their weaker hybridization in comparisonwith specifically bound molecules. More rapid, but less specific, is thedot blot method, which is performed as the Northern blot techniqueexcept that the RNA is directly dotted onto the membrane withoutpreceding fractionation.

A specific method for detection of an mRNA species is the RNaseprotection assay. Total RNA from a tissue or cell culture is hybridizedto a ribonucleotide or deoxyribonucleotide tagged probe, Specificity isaccomplished by subsequent RNase digestion. Non-hybridized,single-stranded RNA and non-specifically hybridized fragments with evensmall mismatches will be recognized and cleaved, while double-strandedRNA or DNA/RNA duplexes of complete homology is not accessible to theenzyme and will be protected. The specific protected fragment can beseparated from degradation products, the tag(s) cleaved from therespective probe and subsequently measured.

The precise location of a given mRNA (or any nucleic acid sequence) in aspecific population of cells within a tissue can be determined by insitu hybridization. In situ hybridization can be even more sensitivethan analysis of a total tissue RNA preparation using the techniquesdescribed above. This is the case when the mRNA is expressed in highconcentrations in a very discrete region or cell type within the tissueand would be diluted by homogenization of the whole tissue. For in situhybridization, the tissues have to be frozen or perfusion-fixed andsectioned according to histochemical protocol. The hybridizationprotocol for tissue sections and the labeled probes used are similar tothe other hybridization methods described above. A quantitative analysisis possible.

10. Mutation Detection Techniques

The detection of diseases is increasingly important in prevention andtreatments. While multifactorial diseases are difficult to devisegenetic tests for, more than 200 known human disorders are caused by adefect in a single gene, often a change of a single amino acid residue(Olsen, Biotechnology: An industry comes of age, National AcademicPress, 1986). Many of these mutations result in an altered amino acidthat causes a disease state.

Sensitive mutation detection techniques offer extraordinarypossibilities for mutation screening. For example, analyses may beperformed even before the implantation of a fertilized egg (Holding andMonk, Lancet 3:532, 1989). Increasingly efficient genetic tests may alsoenable screening for oncogenic mutations in cells exfoliated from therespiratory tract or the bladder in connection with health checkups(Sidransky et al., Science 252:706, 1991). Also, when an unknown genecauses a genetic disease, methods to monitor DNA sequence variants areuseful to study the inheritance of disease through genetic linkageanalysis. However, detecting and diagnosing mutations in individualgenes poses technological and economic challenges. Several differentapproaches have been pursued, but none are both efficient andinexpensive enough for truly widescale application.

Mutations involving a single nucleotide can be identified in a sample byphysical, chemical, or enzymatic means. Generally, methods for mutationdetection may be divided into scanning techniques, which are suitable toidentify previously unknown mutations, and techniques designed todetect, distinguish, or quantitate known sequence variants.

Several scanning techniques for mutation detection have been developedin heteroduplexes of mismatched complementary DNA strands, derived fromwild-type and mutant sequences, exhibit an abnormal behavior especiallywhen denatured. This phenomenon is exploited in denaturing andtemperature gradient gel electrophoresis (DGGE and TGGE, respectively)methods. Duplexes mismatched in even a single nucleotide position canpartially denature, resulting in retarded migration, whenelectrophoresed in an increasingly denaturing gradient gel (Myers etal., Nature 313:495, 1985; Abrams et al., Genomics 7:463, 1990; Henco etal., Nucl. Acids Res. 18:6733, 1990). Although mutations may bedetected, no information is obtained regarding the precise location of amutation. Mutant forms must be further isolated and subjected to DNAsequence analysis.

Alternatively, a heteroduplex of an RNA probe and a target strand may becleaved by RNase A at a position where the two strands are not properlypaired. The site of cleavage can then be determined by electrophoresisof the denatured probe. However, some mutations may escape detectionbecause not all mismatches are efficiently cleaved by RNase A.

Mismatched bases in a duplex are also susceptible to chemicalmodification. Such modification can render the strands susceptible tocleavage at the site of the mismatch or cause a polymerase to stop in asubsequent extension reaction. The chemical cleavage technique allowsidentification of a mutation in target sequences of up to 2 kb and itprovides information on the approximate location of mismatchednucleotide(s) (Cotton et al., PNAS USA 85:4397, 1988; Ganguly et al.,Nucl. Acids Res. 18:3933, 1991). However, this technique is laborintensive and may not identify the precise location of the mutation.

An alternative strategy for detecting a mutation in a DNA strand is bysubstituting (during synthesis) one of the normal nucleotides with amodified nucleotide, altering the molecular weight or other physicalparameter of the product. A strand with an increased or decreased numberof this modified nucleotide relative to the wild-type sequence exhibitsaltered electrophoretic mobility (Naylor et al., Lancet 337:635, 1991).This technique detects the presence of a mutation, but does not providethe location.

Two other strategies visualize mutations in a DNA segment by altered gelmigration. In the single-strand conformation polymorphism technique(SSCP), mutations cause denatured strands to adopt different secondarystructures, thereby influencing mobility during native gelelectrophoresis. Heteroduplex DNA molecules, containing internalmismatches, can also be separated from correctly matched molecules byelectrophoresis (Orita, Genomics 5:874, 1989; Keen, Trends Genet. 7:5,1991). As with the techniques discussed above, the presence of amutation may be determined but not the location. As well, many of thesetechniques do not distinguish between a single and multiple mutations.

All of the above-mentioned techniques indicate the presence of amutation in a limited segment of DNA and some of them allow approximatelocalization within the segment. However, sequence analysis is stillrequired to unravel the effect of the mutation on the coding potentialof the segment. Sequence analysis is very powerful, allowing for examplescreening for the same mutation in other individuals of an affectedfamily, monitoring disease progression in the case of malignant diseaseor for detecting residual malignant cells in the bone marrow beforeautologous transplantation. Despite these advantages, the procedure isunlikely to be adopted as a routine diagnostic method because of thehigh expense involved.

A large number of other techniques have been developed to analyze knownsequence variants. Automation and economy are very importantconsiderations for these types of analyses that may be applied, forscreening individuals and the general population. None of the techniquesdiscussed below combine economy, automation with the requiredspecificity.

Mutations may be identified via their destabilizing effects on thehybridization of short oligonucleotide probes to a target sequence (seeWetmur, Crit. Rev. Biochem. Mol. Biol., 26:227, 1991). Generally, thistechnique, allele-specific oligonucleotide hybridization involvesamplification of target sequences and subsequent hybridization withshort oligonucleotide probes. An amplified product can thus be scannedfor many possible sequence variants by determining its hybridizationpattern to an array of immobilized oligonucleotide probes.

However, establishing conditions that distinguish a number of otherstrategies for nucleotide sequence distinction all depend on enzymes toidentify sequence differences (Saiki, PNAS USA 86:6230, 1989; Zhang,Nucl. Acids Res. 19:3929, 1991).

For example, restriction enzymes recognize sequences of about 4-8nucleotides. Based on an average G+C content, approximately half of thenucleotide positions in a DNA segment can be monitored with a panel of100 restriction enzymes. As an alternative, artificial restrictionenzyme recognition sequences may be created around a variable positionby using partially mismatched PCR primers. With this technique, eitherthe mutant or the wild-type sequence alone may be recognized and cleavedby a restriction enzyme after amplification (Chen et al., Anal. Biochem.195:51, 1991; Levi et al., Cancer Res. 51:3497, 1991).

Another method exploits the property that an oligonucleotide primer thatis mismatched to a target sequence at the 3' penultimate positionexhibits a reduced capacity to serve as a primer in PCR. 1However, some3' mismatches, notably G-T, are less inhibitory than others limiting itsusefulness. In attempts to improve this technique, additional mismatchesare incorporated into the primer at the third position from the 3' end.This results in two mismatched positions in the three 3' nucleotides ofthe primer hybridizing with one allelic variant, and one mismatch in thethird position in from the 3' end when the primer hybridizes to theother allelic variant (Newton et al., Nucl. Acids Res. 17:2503, 1989).It is necessary to define amplification conditions that significantlyfavor amplification of a 1 bp mismatch.

DNA polymerases have also been used to distinguish allelic sequencevariants by determining which nucleotide is added to an oligonucleotideprimer immediately upstream of a variable position in the target strand.

A ligation assay has been developed. In this method, two oligonucleotideprobes hybridizing in immediate juxtaposition on a target strand arejoined by a DNA ligase. Ligation is inhibited if there is a mismatchwhere the two oligonucleotide probes abut.

a. Assays for Mutation Detection

Mutations are a single-base pair change in genomic DNA. Within thecontext of this invention, most such changes are readily detected byhybridization with oligonucleotides that are complementary to thesequence in question. In the system described here, two oligonucleotidesare employed to detect a mutation. One oligonucleotide possesses thewild-type sequence and the other oligonucleotide possesses the mutantsequence. When the two oligonucleotides are used as probes on awild-type target genomic sequence, the wild-type oligonucleotide willform a perfectly based paired structure and the mutant oligonucleotidesequence will form a duplex with a single base pair mismatch.

As discussed above, a 6 to 7° C. difference in the T_(m) of a wild typeversus mismatched duplex permits the ready identification ordiscrimination of the two types of duplexes. To effect thisdiscrimination, hybridization is performed at the T_(m) of themismatched duplex in the respective hybotropic solution. The extent ofhybridization is then measured for the set of oligonucleotide probes.When the ratio of the extent of hybridization of the wild-type probe tothe mismatched probe is measured, a value to 10/1 to greater than 20/1is obtained. These types of results permit the development of robustassays for mutation detection.

For exemplary purposes, one assay format for mutation detection utilizestarget nucleic acid (e.g., genomic DNA) and oligonucleotide probes thatspan the area of interest. The oligonucleotide probes are greater orequal to 24 nt in length (with a maximum of about 36 nt) and labeledwith a fluorochrome at the 3' or 5' end of the oligonucleotide probe.The target nucleic acid is obtained via the lysis of tissue culturecells, tissues, organisms, etc., in the respective hybridizationsolution. The lysed solution is then heated to a temperature whichdenatures the target nucleic acid (15-25° C. above the T_(m) of thetarget nucleic acid duplex). The oligonucleotide probes are added at thedenaturation temperature, and hybridization is conducted at the T_(m) ofthe mismatched duplex for 0.5 to 24 hours. The genomic DNA is thencollected and by passage through a GF/C (GF/B, and the like) glass fiberfilter. The filter is then washed with the respective hybridizationsolution to remove any non-hybridized oligonucleotide probes (RNA, shortoligos and nucleic acid does not bind to glass fiber filters under theseconditions). The hybridization oligo probe can then be thermally elutedfrom the target DNA and measured (by fluorescence for example). Forassays requiring very high levels of sensitivity, the probes areconcentrated and measured.

Other highly sensitive hybridization protocols may be used. The methodsof the present invention enable one to readily assay for a nucleic acidcontaining a mutation suspected of being present in cells, samples,etc., i.e., a target nucleic acid. The "target nucleic acid" containsthe nucleotide sequence of deoxyribonucleic acid (DNA) or ribonucleicacid (RNA) whose presence is of interest, and whose presence or absenceis to be detected for in the hybridization assay. The hybridizationmethods of the present invention may also be applied to a complexbiological mixture of nucleic acid (RNA and/or DNA). Such a complexbiological mixture includes a wide range of eucaryotic and procaryoticcells, including protoplasts; and/or other biological materials whichharbor polynucleotide nucleic acid. The method is thus applicable totissue culture cells, animal cells, animal tissue, blood cells (e.g.,reticulocytes, lymphocytes), plant cells, bacteria, yeasts, viruses,mycoplasmas, protozoa, fungi and the like. By detecting a specifichybridization between nucleic acid probes of a known source, thespecific presence of a target nucleic acid can be established.

A typical hybridization assay protocol for detecting a target nucleicacid in a complex population of nucleic acids is described as follows:Target nucleic acids are separated by size on a gel matrix(electrophoresis), cloned and isolated, sub-divided into pools, or leftas a complex population. The target nucleic acids are transferred,spotted, or immobilized onto a solid support such as a nylon membrane ornitrocellulose membrane. (This "immobilization" is also referred to as"arraying"). The immobilized nucleic acids are then subjected to aheating step or UV radiation, which irreversibly immobilizes the nucleicacid. The membranes are then immersed in "blocking agents" which includeDendhart's reagent (Dendhart, Biochem. Biophys. Res. Comm. 23:641,1966), heparin (Singh and Jones, Nucleic Acids Res. 12:5627, 1984), andnon-fat dried milk (Jones et al., Gene Anal. Tech. 1:3, 1984). Blockingagents are generally included in both the prehybridization step andhybridization steps when nitrocellulose is used. The target nucleicacids are then probed with tagged oligonucleotide probes underconditions described above in hybotrope-based solutions. Unbound enzymeis then washed away and the membrane is immersed in a substratesolution. Signal is then detected by MALD1-MS essentially as describedbelow.

b. Sequencing by hybridization

DNA sequence analysis is conventionally performed by hybridizing aprimer to target DNA and performing chain extensions using a polymerase.Specific stops are controlled by the inclusion of a dideoxynucleotide.The specificity of priming in this type of analysis can be increased byincluding a hybotrope in the annealing buffer and/or incorporating anabasic residue in the primer and annealing at a discriminatingtemperature.

Other sequence analysis methods involve hybridization of the target withan assortment of random, short oligonucleotides. The sequence isconstructed by overlap hybridization analysis. In this technique,precise hybridization is essential. Use of hybotropes or abasic residuesand annealing at a discriminating temperature is beneficial for thistechnique to reduce or eliminate mismatched hybridization. The goal isto develop automated hybridization methods in order to probe largearrays of oligonucleotide probes or large arrays of nucleic acidsamples. Application of such technologies include gene mapping, clonecharacterization, medical genetics and gene discovery, DNA sequenceanalysis by hybridization, and finally, sequencing verification.

Many parameters must be controlled in order to automate or multiplexoligonucleotide probes. The stability of the respective probes must besimilar, the degree of mismatch with the target nucleic acid, thetemperature, ionic strength, the A+T content of the probe (or target),as well as other parameters when the probe is short (i.e., 6 to 50nucleotides) should be similar. Usually, the conditions of theexperiment and the sequence of the probe are adjusted until theformation of the perfectly based paired probe is thermodynamicallyfavored over the any duplex which contains a mismatch. Very large scaleapplications of probes such as sequencing by hybridization (SBH), ortesting highly polymorphic loci such as the cystic fibrosistrans-membrane protein locus require a more stringent level of controlof multiplexed probes.

11. Arrays

Nucleic acid hybridization to arrayed DNA samples has long been employedfor a wide variety of applications in basic biological research, and arecurrently beginning to be used in medical diagnostics, forensics andagriculture. As described in more detail below, nucleic acid moleculesor proteins may be attached to a solid support to form an array, andtested with tagged molecules of the present invention.

For example, within one embodiment of the invention, arrayed DNA samplescan be utilized in the identification of individual clones. Briefly,known DNA molecules are tagged to make a tagged probe, and tested byhybridization against an array of unknown clones. Clones which showspecific hybridization to the probe may then be isolated. Such assaysmay be accomplished using unordered arrays of clones (Sambrook et al.,"Molecular Cloning: A Laboratory Manual" Cold Spring Harbor, N.Y.,1989). Alternatively, membranes carrying regularly spaced arrays ofclones of known individual identity (although typically of unknownsequence) may also be purchased (e.g., Research Genetics, BAC clonearrays, Huntsville, Ala.).

Within another embodiment, arrays may be utilized to measure thetranscription levels of a large number of genes simultaneously (seegenerally, Gess et al., Mammalian Genome 3: 609-619, 1992). Briefly,pools of cDNA may be tagged an utilized as probes on large arrays ofcDNA clones to identify the genes expressed abundantly in specifictissues. Microarrays from individual cDNA clones may also be utilized toquantitatively measure the relative expression of each gene in the arrayin two different RNA samples (Schena et al., Science 270: 467-470, 1995.More specifically, robots may be utilized to produce microarrays of PCRproducts from individual clones: each element in the array correspondsto a single cDNA clone. Probes for the arrays are prepared by labelingfirst strand cDNA from each tissue sample with a tag. To compare geneexpression in two tissue samples, cDNA from each is labelled with adifferent tag. The two samples are pooled and hybridized to the arraytogether. After hybridization of the probes to the array, tags may becleaved and analyzed as described within the present application foreach tag hybridized to each sample in the array. For a given gene, theratio of hybridization to each labeled complex cDNA sample is a measureof the relative gene expression in the two tissue samples. The use ofinternal controls and of two (and potentially up to four) distinct tagsis crucial for this application.

Many of the other applications described below are variations on thisbasic experiment using different sources of arrayed DNA and differentsources of probe DNA, but each application is limited by the use ofconventional detection methods to fewer than 4-6 distinguishable probesin the hybridization mix.

Another application of hybridization to DNA arrays which has beendemonstrated in principle and has the potential for very wideapplication is sequencing by hybridization (SBH). The concept ofsequencing by hybridization (SBH) makes use of an array of all possiblen-nucleotide oligomers (n-mers) to identify n-mers present in an unknownDNA sequence. Computational approaches can then be used to assemble thecomplete sequence (see generally, Drmanac et al., Science 260:1649-1652, 1993). Applications of SBH include physical mapping(ordering) of overlapping DNA clones, sequence checking, DNAfingerprinting comparisons of normal and disease-causing genes, and theidentification of DNA fragments with particular sequence motifs incomplementary DNA and genomic libraries.

DNA arrays also have wide application in the detection of geneticvariations and polymorphisms. Single base pair changes, deletions andinsertions, mutations and polymorphisms can be detected by immobilizingknown sequence variants and probing with labeled PCR products frompatients or pathogens (see, e.g., Guo, et al., Nucleic Acids Res. 22:5456-5465, 1994). Likewise, arrays of oligonucleotides may be utilizedto measure genetic variation, including the detection of drug resistantand drug sensitive variants of HIV (see, e.g., Lipshutz et al.,Biotechniques 19: 442-447, 1995).

DNA arrays can be produced using at least two different techniques:synthesis in situ and deposition of samples produced separately(spotting). One of the most prominent techniques for production of theDNA samples in situ is the light-directed synthesis of oligonucleotidesdescribed in Pease et al, P.N.A.S. USA 91: 5022-6, 1994. Briefly, arraysof defined DNA sequences are produced by the use of photolabile blockinggroups to direct oligonucleotide synthesis in an array using modemphotolithographic methods. Masks are prepared such each array elementthat needs a particular base in the next synthesis step is and exposedto light. A single nucleotide residue is added to each chain that wasexposed by the mask, the synthesis cycle finished, the next cycleinitiated by the use of another mask and another oligonucleotideresidue. Sequential application of this protocol can be used to quicklybuild up very large arrays of oligonucleotides. One version of roboticdeposition is described in Schena et al. (1995) in connection with theuse of arrays for transcription analysis.

Within one embodiment of the invention, second members are arrayed on asolid support such as silica, quartz or glass. The array may then betreated to block non-specific hybridization, followed by incubation offirst member labeled probes on the solid support. Within certainpreferred embodiments the array is then washed with a solution (at adefined stringency) in order to remove non-specifically hybridizingnucleic acids, rinsed with a solution which includes a matrix materialappropriate for spectrometry or potentiometry (e.g., for matrix-assistedlaser desorption and ionization mass spectrometry), dried to form anappropriate matrix, and exposed to light in order to cleave tags fromthe nucleic acid probes. The cleaved tags may then be analyzed byspectrometric or potentiometric techniques (e.g., MALDI-MS).

Within certain embodiments, cleavage and laser desorption occur in asingle step. In other variations, laser desorption and ionization isperformed without a matrix. In some experiments, reference-taggedoligonucleotides or other tagged compounds are added to the matrixsolution to control for variations in the efficiency of photo-cleavage,laser desorption and MS detection efficiency. By measuring the ratio ofabundance between a test tag and a series of reference tags,quantitative information is extracted from the MALDI-MS data.

Within other embodiments the array is composed of oligonucleotides ofless than 50 bp in length. This can be utilized to detect polymorphisms(e.g., single base-pair changes), for genetic mapping, or to detect thepresence or absence of a particular DNA in a sample, for analyzing orsorting clones, paternity testing, foresics, an genetic mapping. Arraysmay likewise be composed of proteins.

Separation of Nucleic Acid Fragments

A sample that requires analysis is often a mixture of many components ina complex matrix. For samples containing unknown compounds, thecomponents must be separated from each other so that each individualcomponent can be identified by other analytical methods. The separationproperties of the components in a mixture are constant under constantconditions, and therefore once determined they can be used to identifyand quantify each of the components. Such procedures are typical inchromatographic and electrophoretic analytical separations.

12. High-Performance Liquid Chromatography (HPLC)

High-Performance liquid chromatography (HPLC) is a chromatographicseparations technique to separate compounds that are dissolved insolution. HPLC instruments consist of a reservoir of mobile phase, apump, an injector, a separation column, and a detector. Compounds areseparated by injecting an aliquot of the sample mixture onto the column.The different components in the mixture pass through the column atdifferent rates due to differences in their partitioning behaviorbetween the mobile liquid phase and the stationary phase.

Recently, IP-RO-HPLC on non-porous PS/DVB particles with chemicallybonded alkyl chains have been shown to be rapid alternatives tocapillary electrophoresis in the analysis of both single anddouble-strand nucleic acids providing similair degrees of resolution(Huber et al, 1993, Anal. Biochem., 212, p351; Huber et al., 1993, Nuc.Acids Res., 21, p1061; Huber et al., 1993, Biotechniques, 16, p898). Incontrast to ion-excahnge chromoatrography, which does not always retaindouble-strand DNA as a function of strand length (Since AT base pairsintereact with the positively charged stationary phase, more stronglythan GC base-pairs), IP-RP-HPLC enables a strictly size-dependentseparation.

A method has been developed using 100 mM triethylammonium acetate asion-pairing reagent, phosphodiester oligonucleotides could besuccessfully separated on alkylated non-porous 2.3 μMpoly(styrene-divinylbenzene) particles by means of high performanceliquid chromatography (Oefner et al., 1994, Anal. Biochem., 223, p39).The technique described allowed the separation of PCR products differingonly 4 to 8 base pairs in length within a size range of 50 to 200nucleotides.

13. Electrophoresis

Electrophoresis is a separations technique that is based on the mobilityof ions (or DNA as is the case described herein) in an electric field.Negatively charged DNA charged migrate towards a positive electrode andpositively-charged ions migrate toward a negative electrode. For safetyreasons one electrode is usually at ground and the other is biasedpositively or negatively. Charged species have different migration ratesdepending on their total charge, size, and shape, and can therefore beseparated. An electrode apparatus consists of a high-voltage powersupply, electrodes, buffer, and a support for the buffer such as apolyacrylamide gel, or a capillary tube. Open capillary tubes are usedfor many types of samples and the other gel supports are usually usedfor biological samples such as protein mixtures or DNA fragments.

14. Capillary Electrophoresis (CE)

Capillary electrophoresis (CE) in its various manifestations (freesolution, isotachophoresis, isoelectric focusing, polyacrylamide gel,micellar electrokinetic "chromatography") is developing as a method forrapid high resolution separations of very small sample volumes ofcomplex mixtures. In combination with the inherent sensitivity andselectivity of MS, CE-MS is a potential powerful technique forbioanalysis. In the novel application disclosed herein, the interfacingof these two methods will lead to superior DNA sequencing methods thateclipse the current rate methods of sequencing by several orders ofmagnitude.

The correspondence between CE and electrospray ionization (ESI) flowrates and the fact that both are facilitated by (and primarily used for)ionic species in solution provide the basis for an extremely attractivecombination. The combination of both capillary zone electrophoresis(CZE) and capillary isotachophoresis with quadrapole mass spectrometersbased upon ESI have been described (Olivares et al., Anal. Chem.59:1230, 1987; Smith et al., Anal. Chem. 60:436, 1988; Loo et al., Anal.Chem. 179:404, 1989; Edmonds et al., J. Chroma. 474:21, 1989; Loo etal., J. Microcolumn Sep. 1:223, 1989; Lee et al., J. Chromatog. 458:313,1988; Smith et al., J. Chromatog. 480:211, 1989; Grese et al., J. Am.Chem. Soc. 111:2835, 1989). Small peptides are easily amenable to CZEanalysis with good (femtomole) sensitivity.

The most powerful separation method for DNA fragments is polyacrylamidegel electrophoresis (PAGE), generally in a slab gel format. However, themajor limitation of the current technology is the relatively long timerequired to perform the gel electrophoresis of DNA fragments produced inthe sequencing reactions. An increase magnitude (10-fold) can beachieved with the use of capillary electrophoresis which utilizeultrathin gels. In free solution to a first approximation all DNAmigrate with the same mobility as the addition of a base results in thecompensation of mass and charge. In polyacrylamide gels, DNA fragmentssieve and migrate as a function of length and this approach has now beenapplied to CE. Remarkable plate number per meter has now been achievedwith cross-linked polyacrylamide (10⁺⁷ plates per meter, Cohen et al.,Proc. Natl. Acad Sci., USA 85:9660, 1988). Such CE columns as describedcan be employed for DNA sequencing. The method of CE is in principle 25times faster than slab gel electrophoresis in a standard sequencer. Forexample, about 300 bases can be read per hour. The separation speed islimited in slab gel electrophoresis by the magnitude of the electricfield which can be applied to the gel without excessive heat production.Therefore, the greater speed of CE is achieved through the use of higherfield strengths (300 V/cm in CE versus 10 V/cm in slab gelelectrophoresis). The capillary format reduces the amperage and thuspower and the resultant heat generation.

Smith and others (Smith et al., Nuc. Acids. Res 18:4417, 1990) havesuggested employing multiple capillaries in parallel to increasethroughput. Likewise, Mathies and Huang (Mathies and Huang, Nature359:167, 1992) have introduced capillary electrophoresis in whichseparations are performed on a parallel array of capillaries anddemonstrated high through-put sequencing (Huang et al., Anal. Chem.64:967, 1992, Huang et al., Anal. Chem. 64:2149, 1992). The majordisadvantage of capillary electrophoresis is the limited amount ofsample that can be loaded onto the capillary. By concentrating a largeamount of sample at the beginning of the capillary, prior to separation,loadability is increased, and detection levels can be lowered severalorders of magnitude. The most popular method of preconcentration in CEis sample stacking. Sample stacking has recently been reviewed (Chienand Burgi, Anal. Chem. 64:489A, 1992). Sample stacking depends of thematrix difference, (pH, ionic strength) between the sample buffer andthe capillary buffer, so that the electric field across the sample zoneis more than in the capillary region. In sample stacking, a large volumeof sample in a low concentration buffer is introduced forpreconcentration at the head of the capillary column. The capillary isfilled with a buffer of the same composition, but at higherconcentration. When the sample ions reach the capillary buffer and thelower electric field, they stack into a concentrated zone. Samplestacking has increased detectabilities 1-3 orders of magnitude.

Another method of preconcentration is to apply isotachophoresis (ITP)prior to the free zone CE separation of analytes. ITP is anelectrophoretic technique which allows microliter volumes of sample tobe loaded on to the capillary, in contrast to the low nL injectionvolumes typically associated with CE. The technique relies on insertingthe sample between two buffers (leading and trailing electrolytes) ofhigher and lower mobility respectively, than the analyte. The techniqueis inherently a concentration technique, where the analytes concentrateinto pure zones migrating with the same speed. The technique iscurrently less popular than the stacking methods described above becauseof the need for several choices of leading and trailing electrolytes,and the ability to separate only cationic or anionic species during aseparation process.

The heart of the DNA sequencing process is the remarkably selectiveelectrophoretic separation of DNA or oligonucleotide fragments. It isremarkable because each fragment is resolved and differs by onlynucleotide. Separations of up to 1000 fragments (1000 bp) have beenobtained. A further advantage of sequencing with cleavable tags is asfollows. There is no requirement to use a slab gel format when DNAfragments are separated by polyacrylamide gel electrophoresis whencleavable tags are employed. Since numerous samples are combined (4 to2000) there is no need to run samples in parallel as is the case withcurrent dye-primer or dye-terminator methods (i.e., ABI373 sequencer).Since there is no reason to run parallel lanes, there is no reason touse a slab gel. Therefore, one can employ a tube gel format for theelectrophoretic separation method. Grossman (Grossman et al., Genet.Anal. Tech. Appl. 9:9, 1992) have shown that considerable advantage isgained when a tube gel format is used in place of a slab gel format.This is due to the greater ability to dissipate Joule heat in a tubeformat compared to a slab gel which results in faster run times (by50%), and much higher resolution of high molecular weight DNA fragments(greater than 1000 nt). Long reads are critical in genomic sequencing.Therefore, the use of cleavable tags in sequencing has the additionaladvantage of allowing the user to employ the most efficient andsensitive DNA separation method which also possesses the highestresolution.

15. Microfabricated Devices

Capillary electrophoresis (CE) is a powerful method for DNA sequencing,forensic analysis, PCR product analysis and restriction fragment sizing.CE is far faster than traditional slab PAGE since with capillary gels afar higher potential field can be applied. However, CE has the drawbackof allowing only one sample to be processed per gel. The method combinesthe faster separations times of CE with the ability to analyze multiplesamples in parallel. The underlying concept behind the use ofmicrofabricated devices is the ability to increase the informationdensity in electrophoresis by miniaturizing the lane dimension to about100 micrometers. The electronics industry routinely usesmicrofabrication to make circuits with features of less than one micronin size. The current density of capillary arrays is limited the outsidediameter of the capillary tube. Microfabrication of channels produces ahigher density of arrays. Microfabrication also permits physicalassemblies not possible with glass fibers and links the channelsdirectly to other devices on a chip. Few devices have been constructedon microchips for separation technologies. A gas chromatograph (Terry etal., IEEE Trans. Electron Device, ED-26: 1880, 1979) and a liquidchromatograph (Manz et al., Sens. Actuators B1:249, 1990) have beenfabricated on silicon chips, but these devices have not been widelyused. Several groups have reported separating fluorescent dyes and aminoacids on microfabricated devices (Manz et al., J. Chromatography593:253, 1992, Effenhauser et al., Anal. Chem. 65:2637, 1993). RecentlyWoolley and Mathies (Woolley and Mathies, Proc. Natl. Acad Sci.91:11348, 1994) have shown that photolithography and chemical etchingcan be used to make large numbers of separation channels on glasssubstrates. The channels are filled with hydroxyethyl cellulose (HEC)separation matrices. It was shown that DNA restriction fragments couldbe separated in as little as two minutes.

Cleavage of Tags

As described above, different linker designs will confer cleavability("lability") under different specific physical or chemical conditions.Examples of conditions which serve to cleave various designs of linkerinclude acid, base, oxidation, reduction, fluoride, thiol exchange,photolysis, and enzymatic conditions.

Examples of cleavable linkers that satisfy the general criteria forlinkers listed above will be well known to those in the art and includethose found in the catalog available from Pierce (Rockford, Ill.).Examples include:

ethylene glycobis(succinimidylsuccinate) (EGS), an amine reactivecross-linking reagent which is cleavable by hydroxylamine (1 M at 37° C.for 3-6 hours);

disuccinimidyl tartarate (DST) and sulfo-DST, which are amine reactivecross-linking reagents, cleavable by 0.015 M sodium periodate;

bis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone (BSOCOES) andsulfo-BSOCOES, which are amine reactive cross-linking reagents,cleavable by base (pH 11.6);

1,4-di-[3'-(2'-pyridyldithio(propionamido)]butane (DPDPB), apyridyldithiol crosslinker which is cleavable by thiol exchange orreduction;

N-[4-(p-azidosalicylamido)-butyl]-3'-(2'-pyridydithio)propionamide(APDP), a pyridyldithiol crosslinker which is cleavable by thiolexchange or reduction;

bis-[beta-4-(azidosalicylamido)ethyl]-disulfide, a photoreactivecrosslinker which is cleavable by thiol exchange or reduction;

N-succinimidyl-(4-azidophenyl)-1,3'dithiopropionate (SADP), aphotoreactive crosslinker which is cleavable by thiol exchange orreduction;

sulfosuccinimidyl-2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionate(SAED), a photoreactive crosslinker which is cleavable by thiol exchangeor reduction;

sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3'dithiopropionate(SAND), a photoreactive crosslinker which is cleavable by thiol exchangeor reduction.

Other examples of cleavable linkers and the cleavage conditions that canbe used to release tags are as follows. A silyl linking group can becleaved by fluoride or under acidic conditions. A 3-, 4-, 5-, or6-substituted-2-nitrobenzyloxy or 2-, 3-, 5-, or6-substituted-4-nitrobenzyloxy linking group can be cleaved by a photonsource (photolysis). A 3-, 4-, 5-, or 6-substituted-2-alkoxyphenoxy or2-, 3-, 5-, or 6-substituted-4-alkoxyphenoxy linking group can becleaved by Ce(NH₄)₂ (NO₃)₆ (oxidation). A NCO₂ (urethane) linker can becleaved by hydroxide (base), acid, or LiAlH₄ (reduction). A 3-pentenyl,2-butenyl, or 1-butenyl linking group can be cleaved by O₃ OSO₄ /IO₄ ⁻,or KMnO₄ (oxidation). A 2-[3-, 4-, or 5-substituted-furyl]oxy linkinggroup can be cleaved by O₂, Br₂, MeOH, or acid.

Conditions for the cleavage of other labile linking groups include:t-alkyloxy linking groups can be cleaved by acid; methyl(dialkyl)methoxyor 4-substituted-2-alkyl-1,3-dioxlane-2-yl linking groups can be cleavedby H₃ O⁺ ; 2-silylethoxy linking groups can be cleaved by fluoride oracid; 2-(X)-ethoxy (where X=keto, ester amide, cyano, NO₂, sulfide,sulfoxide, sulfone) linking groups can be cleaved under alkalineconditions; 2-, 3-, 4-, 5-, or 6-substituted-benzyloxy linking groupscan be cleaved by acid or under reductive conditions; 2-butenyloxylinking groups can be cleaved by (Ph₃ P)₃ RhCl(H), 3-, 4-, 5-, or6-substituted-2-bromophenoxy linking groups can be cleaved by Li, Mg, orBuLi; methylthiomethoxy linking groups can be cleaved by Hg²⁺ ;2-(X)-ethyloxy (where X=a halogen) linking groups can be cleaved by Znor Mg; 2-hydroxyethyloxy linking groups can be cleaved by oxidation(e.g., with Pb(OAc)₄).

Preferred linkers are those that are cleaved by acid or photolysis.Several of the acid-labile linkers that have been developed for solidphase peptide synthesis are useful for linking tags to MOIs. Some ofthese linkers are described in a recent review by Lloyd-Williams etal.(Tetrahedron 49:11065-11133, 1993). One useful type of linker is basedupon p-alkoxybenzyl alcohols, of which two, 4-hydroxymethylphenoxyaceticacid and 4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid, arecommercially available from Advanced ChemTech (Louisville, Ky.). Bothlinkers can be attached to a tag via an ester linkage to thebenzylalcohol, and to an amine-containing MOI via an amide linkage tothe carboxylic acid. Tags linked by these molecules are released fromthe MOI with varying concentrations of trifluoroacetic acid. Thecleavage of these linkers results in the liberation of a carboxylic acidon the tag. Acid cleavage of tags attached through related linkers, suchas 2,4-dimethoxy-4'-(carboxymethyloxy)-benzhydrylamine (available fromAdvanced ChemTech in FMOC-protected form), results in liberation of acarboxylic amide on the released tag.

The photolabile linkers useful for this application have also been forthe most part developed for solid phase peptide synthesis (seeLloyd-Williams review). These linkers are usually based on2-nitrobenzylesters or 2-nitrobenzylamides. Two examples of photolabilelinkers that have recently been reported in the literature are4-(4-(1-Fmoc-amino)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid (Holmesand Jones, J. Org. Chem. 60:2318-2319, 1995) and3-(Fmoc-amino)-3-(2-nitrophenyl)propionic acid (Brown et al., MolecularDiversity 1:4-12, 1995). Both linkers can be attached via the carboxylicacid to an amine on the MOI. The attachment of the tag to the linker ismade by forming an amide between a carboxylic acid on the tag and theamine on the linker. Cleavage of photolabile linkers is usuallyperformed with UV light of 350 nm wavelength at intensities and timesknown to those in the art. Examples of commercial sources of instrumentsfor photochemical cleavage are Aura Industries Inc. (Staten Island,N.Y.) and Agrenetics (Wilmington, Mass.). Cleavage of the linkersresults in liberation of a primary amide on the tag. Examples ofphotocleavable linkers include nitrophenyl glycine esters, exo- andendo-2-benzonorborneyl chlorides and methane sulfonates, and3-amino-3(2-nitrophenyl) propionic acid. Examples of enzymatic cleavageinclude esterases which will cleave ester bonds, nucleases which willcleave phosphodiester bonds, proteases which cleave peptide bonds, etc.

Detection of Tags

Detection methods typically rely on the absorption and emission in sometype of spectral field. When atoms or molecules absorb light, theincoming energy excites a quantized structure to a higher energy level.The type of excitation depends on the wavelength of the light. Electronsare promoted to higher orbitals by ultraviolet or visible light,molecular vibrations are excited by infrared light, and rotations areexcited by microwaves. An absorption spectrum is the absorption of lightas a function of wavelength. The spectrum of an atom or molecule dependson its energy level structure. Absorption spectra are useful foridentification of compounds. Specific absorption spectroscopic methodsinclude atomic absorption spectroscopy (AA), infrared spectroscopy (IR),and UV-vis spectroscopy (uv-vis).

Atoms or molecules that are excited to high energy levels can decay tolower levels by emitting radiation. This light emission is calledfluorescence if the transition is between states of the same spin, andphosphorescence if the transition occurs between states of differentspin. The emission intensity of an analyte is linearly proportional toconcentration (at low concentrations), and is useful for quantifying theemitting species. Specific emission spectroscopic methods include atomicemission spectroscopy (AES), atomic fluorescence spectroscopy (AFS),molecular laser-induced fluorescence (LIF), and X-ray fluorescence(XRF).

When electromagnetic radiation passes through matter, most of theradiation continues in its original direction but a small fraction isscattered in other directions. Light that is scattered at the samewavelength as the incoming light is called Rayleigh scattering. Lightthat is scattered in transparent solids due to vibrations (phonons) iscalled Brillouin scattering. Brillouin scattering is typically shiftedby 0.1 to 1 wave number from the incident light. Light that is scattereddue to vibrations in molecules or optical phonons in opaque solids iscalled Raman scattering. Raman scattered light is shifted by as much as4000 wavenumbers from the incident light. Specific scatteringspectroscopic methods include Raman spectroscopy.

IR spectroscopy is the measurement of the wavelength and intensity ofthe absorption of mid-infrared light by a sample. Mid-infrared light(2.5-50 μm, 4000-200 cm⁻¹) is energetic enough to excite molecularvibrations to higher energy levels. The wavelength of IR absorptionbands are characteristic of specific types of chemical bonds and IRspectroscopy is generally most useful for identification of organic andorganometallic molecules.

Near-infrared absorption spectroscopy (NIR) is the measurement of thewavelength and intensity of the absorption of near-infrared light by asample. Near-infrared light spans the 800 nm-2.5 μm (12,500-4000 cm⁻¹)range and is energetic enough to excite overtones and combinations ofmolecular vibrations to higher energy levels. NIR spectroscopy istypically used for quantitative measurement of organic functionalgroups, especially O--H, N--H, and C═O. The components and design of NIRinstrumentation are similar to uv-vis absorption spectrometers. Thelight source is usually a tungsten lamp and the detector is usually aPbS solid-state detector. Sample holders can be glass or quartz andtypical solvents are CCl₄ and CS₂. The convenient instrumentation of NIRspectroscopy makes it suitable for on-line monitoring and processcontrol.

Ultraviolet and Visible Absorption Spectroscopy (uv-vis) spectroscopy isthe measurement of the wavelength and intensity of absorption ofnear-ultraviolet and visible light by a sample. Absorption in the vacuumUV occurs at 100-200 rm; (10⁵ -50,000 cm⁻¹) quartz UV at 200-350 nm;(50,000-28,570 cm⁻¹) and visible at 350-800 nm; (28,570-12,500 cm⁻¹) andis described by the Beer-Lambert-Bouguet law. Ultraviolet and visiblelight are energetic enough to promote outer electrons to higher energylevels. UV-vis spectroscopy can be usually applied to molecules andinorganic ions or complexes in solution. The uv-vis spectra are limitedby the broad features of the spectra. The light source is usually ahydrogen or deuterium lamp for uv measurements and a tungsten lamp forvisible measurements. The wavelengths of these continuous light sourcesare selected with a wavelength separator such as a prism or gratingmonochromator. Spectra are obtained by scanning the wavelength separatorand quantitative measurements can be made from a spectrum or at a singlewavelength.

Mass spectrometers use the difference in the mass-to-charge ratio (m/z)of ionized atoms or molecules to separate them from each other. Massspectrometry is therefore useful for quantitation of atoms or moleculesand also for determining chemical and structural information aboutmolecules. Molecules have distinctive fragmentation patterns thatprovide structural information to identify compounds. The generaloperations of a mass spectrometer are as follows. Gas-phase ions arecreated, the ions are separated in space or time based on theirmass-to-charge ratio, and the quantity of ions of each mass-to-chargeratio is measured. The ion separation power of a mass spectrometer isdescribed by the resolution, which is defined as R=m/delta m, where m isthe ion mass and delta m is the difference in mass between tworesolvable peaks in a mass spectrum. For example, a mass spectrometerwith a resolution of 1000 can resolve an ion with a m/z of 100.0 from anion with a m/z of 100.1.

In general, a mass spectrometer (MS) consists of an ion source, amass-selective analyzer, and an ion detector. The magnetic-sector,quadrupole, and time-of-flight designs also require extraction andacceleration ion optics to transfer ions from the source region into themass analyzer. The details of several mass analyzer designs (formagnetic-sector MS, quadrupole MS or time-of-flight MS) are discussedbelow. Single Focusing analyzers for magnetic-sector MS utilize aparticle beam path of 180, 90, or 60 degrees. The various forcesinfluencing the particle separate ions with different mass-to-chargeratios. With double-focusing analyzers, an electrostatic analyzer isadded in this type of instrument to separate particles with differencein kinetic energies.

A quadrupole mass filter for quadrupole MS consists of four metal rodsarranged in parallel. The applied voltages affect the trajectory of ionstraveling down the flight path centered between the four rods. For givenDC and AC voltages, only ions of a certain mass-to-charge ratio passthrough the quadrupole filter and all other ions are thrown out of theiroriginal path, A mass spectrum is obtained by monitoring the ionspassing through the quadrupole filter as the voltages on the rods arevaried.

A time-of-flight mass spectrometer uses the differences in transit timethrough a "drift region" to separate ions of different masses. Itoperates in a pulsed mode so ions must be produced in pulses and/orextracted in pulses. A pulsed electric field accelerates all ions into afield-free drift region with a kinetic energy of qV, where q is the ioncharge and V is the applied voltage. Since the ion kinetic energy is 0.5mV², lighter ions have a higher velocity than heavier ions and reach thedetector at the end of the drift region sooner. The output of an iondetector is displayed on an oscilloscope as a function of time toproduce the mass spectrum.

The ion formation process is the starting point for mass spectrometricanalyses. Chemical ionization is a method that employs a reagent ion toreact with the analyte molecules (tags) to form ions by either a protonor hydride transfer. The reagent ions are produced by introducing alarge excess of methane (relative to the tag) into an electron impact(EI) ion source. Electron collisions produce CH₄ ⁺ 0 and CH₃ ⁺ whichfurther react with methane to form CH₅ ⁺ and C₂ H⁵ ⁺. Another method toionize tags is by plasma and glow discharge. Plasma is a hot,partially-ionized gas that effectively excites and ionizes atoms. A glowdischarge is a low-pressure plasma maintained between two electrodes.Electron impact ionization employs an electron beam, usually generatedfrom a tungsten filament, to ionize gas-phase atoms or molecules. Anelectron from the beam knocks an electron off analyte atoms or moleculesto create ions. Electrospray ionization utilizes a very fine needle anda series of skimmers. A sample solution is sprayed into the sourcechamber to form droplets. The droplets carry charge when the exit thecapillary and as the solvent vaporizes the droplets disappear leavinghighly charged analyte molecules. ESI is particularly useful for largebiological molecules that are difficult to vaporize or ionize. Fast-atombombardment (FAB) utilizes a high-energy beam of neutral atoms,typically Xe or Ar, that strikes a solid sample causing desorption andionization. It is used for large biological molecules that are difficultto get into the gas phase. FAB causes little fragmentation and usuallygives a large molecular ion peak, making it useful for molecular weightdetermination. The atomic beam is produced by accelerating ions from anion source though a charge-exchange cell. The ions pick up an electronin collisions with neutral atoms to form a beam of high energy atoms.Laser ionization (LIMS) is a method in which a laser pulse ablatesmaterial from the surface of a sample and creates a microplasma thationizes some of the sample constituents. Matrix-assisted laserdesorption ionization (MALDI) is a LIMS method of vaporizing andionizing large biological molecules such as proteins or DNA fragments.The biological molecules are dispersed in a solid matrix such asnicotinic acid. A UV laser pulse ablates the matrix which carries someof the large molecules into the gas phase in an ionized form so they canbe extracted into a mass spectrometer. Plasma-desorption ionization (PD)utilizes the decay of ²⁵² Cf which produces two fission fragments thattravel in opposite directions. One fragment strikes the sample knockingout 1-10 analyte ions. The other fragment strikes a detector andtriggers the start of data acquisition. This ionization method isespecially useful for large biological molecules. Resonance ionization(RIMS) is a method in which one or more laser beams are tuned inresonance to transitions of a gas-phase atom or molecule to promote itin a stepwise fashion above its ionization potential to create an ion.Secondary ionization (SIMS) utilizes an ion beam; such as ³ He⁺,¹⁶ O⁺,or ⁴⁰ Ar⁺ ; is focused onto the surface of a sample and sputtersmaterial into the gas phase. Spark source is a method which ionizesanalytes in solid samples by pulsing an electric current across twoelectrodes.

A tag may become charged prior to, during or after cleavage from themolecule to which it is attached. Ionization methods based on ion"desorption", the direct formation or emission of ions from solid orliquid surfaces have allowed increasing application to nonvolatile andthermally labile compounds. These methods eliminate the need for neutralmolecule volatilization prior to ionization and generally minimizethermal degradation of the molecular species. These methods includefield desorption (Becky, Principles of Field Ionization and FieldDesorption Mass Spectrometry, Pergamon, Oxford, 1977), plasma desorption(Sundqvist and Macfarlane, Mass Spectrom. Rev. 4:421, 1985), laserdesorption (Karas and Hillenkamp, Anal. Chem. 60:2299, 1988; Karas etal., Angew. Chem. 101:805, 1989), fast particle bombardment (e.g., fastatom bombardment, FAB, and secondary ion mass spectrometry, SIMS, Barberet al., Anal. Chem. 54:645A, 1982), and thermospray (TS) ionization(Vestal, Mass Spectrom. Rev. 2:447, 1983). Thermospray is broadlyapplied for the on-line combination with liquid chromatography. Thecontinuous flow FAB methods (Caprioli et al., Anal. Chem. 58:2949, 1986)have also shown significant potential. A more complete listing ofionization/mass spectrometry combinations is ion-trap mass spectrometry,electrospray ionization mass spectrometry, ion-spray mass spectrometry,liquid ionization mass spectrometry, atmospheric pressure ionizationmass spectrometry, electron ionization mass spectrometry, metastableatom bombardment ionization mass spectrometry, fast atom bombardionization mass spectrometry, MALDI mass spectrometry, photo-ionizationtime-of-flight mass spectrometry, laser droplet mass spectrometry,MALDI-TOF mass spectrometry, APCI mass spectrometry, nano-spray massspectrometry, nebulised spray ionization mass spectrometry, chemicalionization mass spectrometry, resonance ionization mass spectrometry,secondary ionization mass spectrometry, thermospray mass spectrometry.

The ionization methods amenable to nonvolatile biological compounds haveoverlapping ranges of applicability. Ionization efficiencies are highlydependent on matrix composition and compound type. Currently availableresults indicate that the upper molecular mass for TS is about 8000daltons (Jones and Krolik, Rapid Comm. Mass Spectrom. 1:67, 1987). SinceTS is practiced mainly with quadrapole mass spectrometers, sensitivitytypically suffers disporportionately at higher mass-to-charge ratios(m/z). Time-of-flight (TOF) mass spectrometers are commerciallyavailable and possess the advantage that the m/z range is limited onlyby detector efficiency. Recently, two additional ionization methods havebeen introduced. These two methods are now referred to asmatrix-assisted laser desorption (MALDI, Karas and Hillenkamp, Anal.Chem. 60:2299, 1988; Karas et al., Angew. Chem. 101:805, 1989) andelectrospray ionization (ESI). Both methodologies have very highionization efficiency (i.e., very high [molecular ionsproduced]/[molecules consumed]). Sensitivity, which defines the ultimatepotential of the technique, is dependent on sample size, quantity ofions, flow rate, detection efficiency and actual ionization efficiency.

Electrospray-MS is based on an idea first proposed in the 1960s (Dole etal., J. Chem. Phys. 49:2240, 1968). Electrospray ionization (ESI) is onemeans to produce charged molecules for analysis by mass spectroscopy.Briefly, electrospray ionization produces highly charged droplets bynebulizing liquids in a strong electrostatic field. The highly chargeddroplets, generally formed in a dry bath gas at atmospheric pressure,shrink by evaporation of neutral solvent until the charge repulsionovercomes the cohesive forces, leading to a "Coulombic explosion". Theexact mechanism of ionization is controversial and several groups haveput forth hypotheses (Blades et al., Anal. Chem. 63:2109-14, 1991;Kebarle et al., Anal. Chem. 65:A972-86, 1993; Fenn, J. Am. Soc. Mass.Spectrom. 4:524-35, 1993). Regardless of the ultimate process of ionformation, ESI produces charged molecules from solution under mildconditions.

The ability to obtain useful mass spectral data on small amounts of anorganic molecule relies on the efficient production of ions. Theefficiency of ionization for ESI is related to the extent of positivecharge associated with the molecule. Improving ionization experimentallyhas usually involved using acidic conditions. Another method to improveionization has been to use quaternary amines when possible (seeAebersold et al., Protein Science 1:494-503, 1992; Smith et al., Anal.Chem. 60:436-41, 1988).

Electrospray ionization is described in more detail as follows.Electrospray ion production requires two steps: dispersal of highlycharged droplets at near atmospheric pressure, followed by conditions toinduce evaporation. A solution of analyte molecules is passed through aneedle that is kept at high electric potential. At the end of theneedle, the solution disperses into a mist of small highly chargeddroplets containing the analyte molecules. The small droplets evaporatequickly and by a process of field desorption or residual evaporation,protonated protein molecules are released into the gas phase. Anelectrospray is generally produced by application of a high electricfield to a small flow of liquid (generally 1-10 uL/min) from a capillarytube. A potential difference of 3-6 kV is typically applied between thecapillary and counter electrode located 0.2-2 cm away (where ions,charged clusters, and even charged droplets, depending on the extent ofdesolvation, may be sampled by the MS through a small orifice). Theelectric field results in charge accumulation on the liquid surface atthe capillary terminus; thus the liquid flow rate, resistivity, andsurface tension are important factors in droplet production. The highelectric field results in disruption of the liquid surface and formationof highly charged liquid droplets. Positively or negatively chargeddroplets can be produced depending upon the capillary bias. The negativeion mode requires the presence of an electron scavenger such as oxygento inhibit electrical discharge.

A wide range of liquids can be sprayed electrostatically into a vacuum,or with the aid of a nebulizing agent. The use of only electric fieldsfor nebulization leads to some practical restrictions on the range ofliquid conductivity and dielectric constant. Solution conductivity ofless than 10⁻⁵ ohms is required at room temperature for a stableelectrospray at useful liquid flow rates corresponding to an aqueouselectrolyte solution of <10⁻⁴ M. In the mode found most useful forESI-MS, an appropriate liquid flow rate results in dispersion of theliquid as a fine mist. A short distance from the capillary the dropletdiameter is often quite uniform and on the order of 1 μm. Of particularimportance is that the total electrospray ion current increases onlyslightly for higher liquid flow rates. There is evidence that heating isuseful for manipulating the electrospray. For example, slight heatingallows aqueous solutions to be readily electrosprayed, presumably due tothe decreased viscosity and surface tension. Both thermally-assisted andgas-nebulization-assisted electrosprays allow higher liquid flow ratesto be used, but decrease the extent of droplet charging. The formationof molecular ions requires conditions effecting evaporation of theinitial droplet population. This can be accomplished at higher pressuresby a flow of dry gas at moderate temperatures (<60° C.), by heatingduring transport through the interface, and (particularly in the case ofion trapping methods) by energetic collisions at relatively lowpressure.

Although the detailed processes underlying ESI remain uncertain, thevery small droplets produced by ESI appear to allow almost any speciescarrying a net charge in solution to be transferred to the gas phaseafter evaporation of residual solvent. Mass spectrometric detection thenrequires that ions have a tractable m/z range (<4000 daltons forquadrupole instruments) after desolvation, as well as to be produced andtransmitted with sufficient efficiency. The wide range of solutesalready found to be amenable to ESI-MS, and the lack of substantialdependence of ionization efficiency upon molecular weight, suggest ahighly non-discriminating and broadly applicable ionization process.

The electrospray ion "source" functions at near atmospheric pressure.The electrospray "source" is typically a metal or glass capillaryincorporating a method for electrically biasing the liquid solutionrelative to a counter electrode. Solutions, typically water-methanolmixtures containing the analyte and often other additives such as aceticacid, flow to the capillary terminus. An ESI source has been described(Smith et al., Anal. Chem. 62:885, 1990) which can accommodateessentially any solvent system. Typical flow rates for ESI are 1-10uL/min. The principal requirement of an ESI-MS interface is to sampleand transport ions from the high pressure region into the MS asefficiently as possible.

The efficiency of ESI can be very high, providing the basis forextremely sensitive measurements, which is useful for the inventiondescribed herein. Current instrumental performance can provide a totalion current at the detector of about 2×10⁻¹² A or about 10⁷ counts/s forsingly charged species. On the basis of the instrumental performance,concentrations of as low as 10⁻¹⁰ M or about 10⁻¹⁸ mol/s of a singlycharged species will give detectable ion current (about 10 counts/s) ifthe analyte is completely ionized. For example, low attomole detectionlimits have been obtained for quaternary ammonium ions using an ESIinterface with capillary zone electrophoresis (Smith et al., Anal. Chem.59:1230, 1988). For a compound of molecular weight of 1000, the averagenumber of charges is 1, the approximate number of charge states is 1,peak width (m/z) is 1 and the maximum intensity (ion/s) is 1×10¹².

Remarkably little sample is actually consumed in obtaining an ESI massspectrum (Smith et al., Anal. Chem. 60:1948, 1988). Substantial gainsmight be also obtained by the use of array detectors with sectorinstruments, allowing simultaneous detection of portions of thespectrum. Since currently only about 10⁻⁵ of all ions formed by ESI aredetected, attention to the factors limiting instrument performance mayprovide a basis for improved sensitivity. It will be evident to those inthe art that the present invention contemplates and accommodates forimprovements in ionization and detection methodologies.

An interface is preferably placed between the separation instrumentation(e.g., gel)and the detector (e.g., mass spectrometer). The interfacepreferably has the following properties: (1) the ability to collect theDNA fragments at discreet time intervals, (2) concentrate the DNAfragments, (3) remove the DNA fragments from the electrophoresis buffersand milieu, (4) cleave the tag from the DNA fragment, (5) separate thetag from the DNA fragment, (6) dispose of the DNA fragment, (7) placethe tag in a volatile solution, (8) volatilize and ionize the tag, and(9) place or transport the tag to an electrospray device that introducesthe tag into mass spectrometer.

The interface also has the capability of "collecting" DNA fragments asthey elute from the bottom of a gel. The gel may be composed of a slabgel, a tubular gel, a capillary, etc. The DNA fragments can be collectedby several methods. The first method is that of use of an electric fieldwherein DNA fragments are collected onto or near an electrode. A secondmethod is that wherein the DNA fragments are collected by flowing astream of liquid past the bottom of a gel. Aspects of both methods canbe combined wherein DNA collected into a flowing stream which can belater concentrated by use of an electric field. The end result is thatDNA fragments are removed from the milieu under which the separationmethod was performed. That is, DNA fragments can be "dragged" from onesolution type to another by use of an electric field.

Once the DNA fragments are in the appropriate solution (compatible withelectrospray and mass spectrometry) the tag can be cleaved from the DNAfragment. The DNA fragment (or remnants thereof) can then be separatedfrom the tag by the application of an electric field (preferably, thetag is of opposite charge of that of the DNA tag). The tag is thenintroduced into the electrospray device by the use of an electric fieldor a flowing liquid.

Fluorescent tags can be identified and quantitated most directly bytheir absorption and fluorescence emission wavelengths and intensities.

While a conventional spectrofluorometer is extremely flexible, providingcontinuous ranges of excitation and emission wavelengths (I_(EX),I_(S1), I_(S2))l more specialized instruments such as flow cytometersand laser-scanning microscopes require probes that are excitable at asingle fixed wavelength. In contemporary instruments, this is usuallythe 488-nm line of the argon laser.

Fluorescence intensity per probe molecule is proportional to the productof e and QY. The range of these parameters among fluorophores of currentpractical importance is approximately 10,000 to 100,000 cm⁻¹ M⁻¹ for εand 0.1 to 1.0 for QY. When absorption is driven toward saturation byhigh-intensity illumination, the irreversible destruction of the excitedfluorophore (photobleaching) becomes the factor limiting fluorescencedetectability. The practical impact of photobleaching depends on thefluorescent detection technique in question.

It will be evident to one in the art that a device (an interface) may beinterposed between the separation and detection steps to permit thecontinuous operation of size separation and tag detection (in realtime). This unites the separation methodology and instrumentation withthe detection methodology and instrumentation forming a single device.For example, an interface is interposed between a separation techniqueand detection by mass spectrometry or potentiostatic amperometry.

The function of the interface is primarily the release of the (e.g.,mass spectrometry) tag from analyte. There are several representativeimplementations of the interface. The design of the interface isdependent on the choice of cleavable linkers. In the case of light orphoto-cleavable linkers, an energy or photon source is required. In thecase of an acid-labile linker, a base-labile linker, or a disulfidelinker, reagent addition is required within the interface. In the caseof heat-labile linkers, an energy heat source is required. Enzymeaddition is required for an enzyme-sensitive linker such as a specificprotease and a peptide linker, a nuclease and a DNA or RNA linker, aglycosylase, HRP or phosphatase and a linker which is unstable aftercleavage (e.g., similiar to chemiluminescent substrates). Othercharacteristics of the interface include minimal band broadening,separation of DNA from tags before injection into a mass spectrometer.Separation techniques include those based on electrophoretic methods andtechniques, affinity techniques, size retention (dialysis), filtrationand the like.

It is also possible to concentrate the tags (or nucleic acid-linker-tagconstruct), capture electrophoretically, and then release into alternatereagent stream which is compatible with the particular type ofionization method selected. The interface may also be capable ofcapturing the tags (or nucleic acid-linker-tag construct) on microbeads,shooting the bead(s) into chamber and then preforming laserdesorption/vaporization. Also it is possible to extract in flow intoalternate buffer (e.g., from capillary electrophoresis buffer intohydrophobic buffer across a permeable membrane). It may also bedesirable in some uses to deliver tags into the mass spectrometerintermittently which would comprise a further function of the interface.Another function of the interface is to deliver tags from multiplecolumns into a mass spectrometer, with a rotating time slot for eachcolumn. Also, it is possible to deliver tags from a single column intomultiple MS detectors, separated by time, collect each set of tags for afew milliseconds, and then deliver to a mass spectrometer.

The following is a list of representative vendors for separation anddetection technologies which may be used in the present invention.Hoefer Scientific Instruments (San Francisco, Calif.) manufactureselectrophoresis equipment (Two Step™, Poker Face™ II) for sequencingapplications. Pharmacia Biotech (Piscataway, N.J.) manufactureselectrophoresis equipment for DNA separations and sequencing(PhastSystem for PCR-SSCP analysis, MacroPhor System for DNAsequencing). Perkin Elmer/Applied Biosystems Division (ABI, Foster City,Calif.) manufactures semi-automated sequencers based on fluorescent-dyes(ABI373 and ABI377). Analytical Spectral Devices (Boulder, Colo.)manufactures UV spectrometers. Hitachi Instruments (Tokyo, Japan)manufactures Atomic Absorption spectrometers, Fluorescencespectrometers, LC and GC Mass Spectrometers, NMR spectrometers, andUV-VIS Spectrometers. PerSeptive Biosystems (Framingham, Mass.) producesMass Spectrometers (Voyager™ Elite). Bruker Instruments Inc. (ManningPark, Mass.) manufactures FTIR Spectrometers (Vector 22), FT-RamanSpectrometers, Time of Flight Mass Spectrometers (Reflex II™), Ion TrapMass Spectrometer (Esquire™) and a Maldi Mass Spectrometer. AnalyticalTechnology Inc. (ATI, Boston, Mass.) makes Capillary Gel Electrophoresisunits, UV detectors, and Diode Array Detectors. Teledyne ElectronicTechnologies (Mountain View, Calif.) manufactures an Ion Trap MassSpectrometer (3DQ Discovery™ and the 3DQ Apogee™). Perkin Elmer/AppliedBiosystems Division (Foster City, Calif.) manufactures a Sciex MassSpectrometer (triple quadrupole LC/MS/MS, the API 100/300) which iscompatible with clectrospray. Hewlett-Packard (Santa Clara, Calif.)produces Mass Selective Detectors (HP 5972A), MALDI-TOF MassSpectrometers (HP G2025A), Diode Array Detectors, CE units, HPLC units(HP1090) as well as UV Spectrometers. Finnigan Corporation (San Jose,Calif.) manufactures mass spectrometers (magnetic sector (MAT 95 S™),quadrapole spectrometers (MAT 95 SQ™) and four other related massspectrometers). Rainin (Emeryville, Calif.) manufactures HPLCinstruments.

The methods and compositions described herein permit the use of cleavedtags to serve as maps to particular sample type and nucleotide identity.At the beginning of each sequencing method, a particular (selected)primer is assigned a particular unique tag. The tags map to either asample type, a dideoxy terminator type (in the case of a Sangersequencing reaction) or preferably both. Specifically, the tag maps to aprimer type which in turn maps to a vector type which in turn maps to asample identity. The tag may also may map to a dideoxy terminator type(ddTTP, ddCTP, ddGTP, ddATP) by reference into which dideoxynucleotidereaction the tagged primer is placed. The sequencing reaction is thenperformed and the resulting fragments are sequentially separated by sizein time.

The tags are cleaved from the fragments in a temporal frame and measuredand recorded in a temporal frame. The sequence is constructed bycomparing the tag map to the temporal frame. That is, all tag identitiesare recorded in time after the sizing step and related become related toone another in a temporal frame. The sizing step separates the nucleicacid fragments by a one nucleotide increment and hence the related tagidentities are separated by a one nucleotide increment. By foreknowledgeof the dideoxy-terminator or nucleotide map and sample type, thesequence is readily deduced in a linear fashion.

In an embodiment of the present invention, an array interrogation systemis provided that includes a DNA array generating device, a cleavingdevice, a desorpting device, a detecting device and a data processor andanalyzer that analyzes data from the detecting devise to correlate a tagwith a nucleic acid fragment from a sample. As best seen in FIG. 14, thearray interrogation system 10 includes a DNA array generating device 12that provides an arrayed DNA chip 14 with selected samples of nucleicacid fragments and cleavable mass spectrometer tags (CMST) attached tothe nucleic acid fragments. The arrayed DNA chip 14 is passed through orpast a photolytic cleavage device 16 that cleaves the CMSTs from thenucleic acid fragments while still on the DNA chip 14.

After CMSTs are cleaved, the DNA chip 14 is positioned in an automatedmicro-array sampling laser device 18, such as a Matrix Assisted LaserDesorption Ionization (MALDI) instrument. The MALDI instrument 18 isadapted to irradiate the CMSTs and cause desorption of the CMSTs. TheCMSTs, after desorption, are then transferred to a detection device 22,such as a mass spectrometer, wherein the CMSTs are detected based uponthe difference in molecular weight between each of the tags used tolabel the nucleic acid fragment.

Data from the detection device 22 is provided to the data processor andanalyzer 24, which includes a software program that maps the signatureof a given tag to a specific sample ID. The software is able to displaythe DNA sequence determined and load the sequence information intorespective data bases.

In an alternate embodiment (not shown), the MALDI instrument 18 includesan additional light source that is capable of irradiating the entire DNAchip at an a wavelength in the range of 250 to 360 nm with adjustableintensity, so as to cause the photolytic cleaving of the CMSTs.Accordingly, the cleaving device 16 is incorporated as a component ofthe MALDI instrument 18. After cleaving the CMSTs, the MALDI instrument18 volitized the CMSTs, which are transferred to the detecting device 14as discussed above.

In another embodiment (not shown), the DNA chip 14 is moved from the DNAarray generating device 12 directly to the MALDI instrument 18. TheMALDI instrument 18 includes a laser that emits at a wavelength in therange of approximately 250 to 360 nm, inclusive. The laser causes thesimultaneous photolytic cleavage of the tag from the nucleic acidfragment along with simultaneous desorption of the CMST. The CMSTs arethen transferred to the mass spectrometer or other detection device 22as discussed above. Accordingly, this alternate embodiment providesphotocleavage by the MALDI instrument, so a separate cleavage device isnot needed.

Tagged Molecules in Array-Based Assays

Arrays with covalently attached oligonucleotides have been made used toperform DNA sequence analysis by hybridization (Southern et al.,Genomics 13: 1008, 1992; Drmanac et al., Science 260: 1649, 1993),determine expression profiles, screen for mutations and the like. Ingeneral, detection for these assays uses fluorescent or radioactivelabels. Fluorescent labels can be identified and quantitated mostdirectly by their absorption and fluorescence emission wavelengths andintensity. A microscope/camera setup using a fluorescent light source isa convenient means for detecting fluorescent label. Radioactive labelsmay be visualized by standard autoradiography, phosphor image analysisor CCD detector. For such labels the number of different reactions thatcan be detected at a single time is limited. For example, the use offour fluorescent molecules, such as commonly employed in DNA sequenceanalysis, limits anaylsis to four samples at a time. Essentially,because of this limitation, each reaction must be individually assessedwhen using these detector methods.

A more advantageous method of detection allows pooling of the samplereactions on at least one array and simultaneous detection of theproducts. By using a tag, such as the ones described herein, having adifferent molecular weight or other physical attribute in each reaction,the entire set of reaction products can be harvested together andanalyzed.

As noted above, the methods described herein are applicable for avariety of purposes. For example, the arrays of oligonucleotides may beused to control for quality of making arrays, for quantitation orqualitative analysis of nucleic acid molecules, for detecting mutations,for determining expression profiles, for toxicology testing, and thelike.

16. Probe quantitation or typing

In this embodiment, oligonucleotides are immobilized per element in anarray where each oligonucleotide in the element is a different orrelated sequence. Preferably, each element possesses a known or relatedset of sequences. The hybridization of a labeled probe to such an arraypermits the characterization of a probe and the identification andquantification of the sequences contained in a probe population.

A generalized assay format that may be used in the particularapplications discussed below is a sandwich assay format. In this format,a plurality of oligonucleotides of known sequence are immobilized on asolid substrate. The immobilized oligonucleotide is used to capture anucleic acid (e.g., RNA, rRNA, a PCR product, fragmented DNA) and then asignal probe is hybridized to a different portion of the captured targetnucleic acid.

Another generalized assay format is a secondary detection system. Inthis format, the arrays are used to identify and quantify labelednucleic acids that have been used in a primary binding assay. Forexample, if an assay results in a labeled nucleic acid, the identity ofthat nucleic acid can be determined by hybridization to an array. Theseassay formats are particularly useful when combined with cleavable massspectometry tags.

17. Mutation detection

Mutations involving a single nucleotide can be identified in a sample byscanning techniques, which are suitable to identify previously unknownmutations, or by techniques designed to detect, distinguish, orquantitate known sequence variants. Several scanning techniques formutation detection have been developed based on the observation thatheteroduplexes of mismatched complementary DNA strands, derived fromwild type and mutant sequences, exhibit an abnormal migratory behavior.

The methods described herein may be used for mutation screening. Onestrategy for detecting a mutation in a DNA strand is by hybridization ofthe test sequence to target sequences that are wild-type or mutantsequences. A mismatched sequence has a destabilizing effect on thehybridization of short oligonucleotide probes to a target sequence (seeWetmur, Crit. Rev. Biochem. Mol. Biol., 26:227, 1991). The test nucleicacid source can be genomic DNA, RNA, cDNA, or amplification of any ofthese nucleic acids. Preferably, amplification of test sequences isfirst performed, followed by hybridization with short oligonucleotideprobes immobilized on an array. An amplified product can be scanned formany possible sequence variants by determining its hybridization patternto an array of immobilized oligonucleotide probes.

A label, such as described herein, is generally incorporated into thefinal amplification product by using a labeled nucleotide or by using alabeled primer. The amplification product is denatured and hybridized tothe array. Unbound product is washed off and label bound to the array isdetected by one of the methods herein. For example, when cleavable massspectrometry tags are used, multiple products can be simultaneouslydetected.

18. Expression profiles/differential display

Mammals, such as human beings, have about 100,000 different genes intheir genome, of which only a small fraction, perhaps 15%, are expressedin any individual cell. Differential display techniques permit theidentification of genes specific for individual cell types. Briefly, indifferential display, the 3' terminal portions of mRNAs are amplifiedand identified on the basis of size. Using a primer designed to bind tothe 5' boundary of a poly(A) tail for reverse transcription, followed byamplification of the cDNA using upstream arbitrary sequence primers,mRNA sub-populations are obtained.

As disclosed herein, a high throughput method for measuring theexpression of numerous genes (e.g., 1-2000) is provided. Within oneembodiment of the invention, methods are provided for analyzing thepattern of gene expression from a selected biological sample, comprisingthe steps of (a) amplifying cDNA from a biological sample using one ormore tagged primers, wherein the tag is correlative with a particularnucleic acid probe and detectable by non-fluorescent spectrometry orpotentiometry, (b) hybridizing amplified fragments to an array ofoligonucleotides as described herein, (c) washing away non-hybridizedmaterial, and (d) detecting the tag by non-fluorescent spectrometry orpotentiometry, and therefrom determining the pattern of gene expressionof the biological sample. Tag-based differential display, especiallyusing cleavable mass spectometry tags, on solid substrates allowscharacterization of differentially expressed genes.

19. Single nucleotide extension assay

The primer extension technique may be used for the detection of singlenucleotide changes in a nucleic acid template (Sokolov, Nucleic AcidsRes., 18:3671, 1989). The technique is generally applicable to detectionof any single base mutation (Kuppuswamy et al., Proc. Natl, Acad. Sci.USA, 88:1143-1147, 1991). Briefly, this method first hybridizes a primerto a sequence adjacent to a known single nucleotide polymorphism. Theprimed DNA is then subjected to conditions in which a DNA polymeraseadds a labeled dNTP, typically a ddNTP, if the next base in the templateis complementary to the labeled nucleotide in the reaction mixture. In amodification, cDNA is first amplified for a sequence of interestcontaining a single-base difference between two alleles. Each amplifiedproduct is then analyzed for the presence, absence, or relative amountsof each allele by annealing a primer that is 1 base 5' to thepolymorphism and extending by one labeled base (generally adideoxynucleotide). Only when the correct base is available in thereaction will a base to incorporated at the 3'-end of the primer.Extension products are then analyzed by hybridization to an array ofoligonucleotides such that a non-extended product will not hybridize.

Briefly, in the present invention, each dideoxynucleotide is labeledwith a unique tag. Of the four reaction mixtures, only one will add adideoxy-terminator on to the primer sequence. If the mutation ispresent, it will be detected through the unique tag on thedideoxynucleotide after hybridization to the array. Multiple mutationscan be simultaneously determined by tagging the DNA primer with a uniquetag as well. Thus, the DNA fragments are reacted in four separatereactions each including a different tagged dideoxyterminator, whereinthe tag is correlative with a particular dideoxynucleotide anddetectable by non-fluorescent spectrometry, or potentiometry. The DNAfragments are hybridized to an array and non-hybridized material iswashed away. The tags are cleaved from the hybridized fragments anddetected by the respective detection technology (e.g., massspectrometry, infrared spectrometry, potentiostatic amperometry orUV/visible spectrophotometry). The tags detected can be correlated tothe particular DNA fragment under investigation as well as the identityof the mutant nucleotide.

20. Oligonucleotide ligation assay

The oligonucleotide ligation assay (OLA). (Landegen et al., Science241:487, 1988) is used for the identification of known sequences in verylarge and complex genomes. The principle of OLA is based on the abilityof ligase to covalently join two diagnostic oligonucleotides as theyhybridize adjacent to one another on a given DNA target. If thesequences at the probe junctions are not perfectly based-paired, theprobes will not be joined by the ligase. When tags are used, they areattached to the probe, which is ligated to the amplified product. Aftercompletion of OLA, fragments are hybridized to an array of complementarysequences, the tags cleaved and detected by mass spectrometry.

Within one embodiment of the invention methods are provided fordetermining the identity of a nucleic acid molecule, or for detecting aselecting nucleic acid molecule, in, for example a biological sample,utilizing the technique of oligonucleotide ligation assay. Briefly, suchmethods generally comprise the steps of performing amplification on thetarget DNA followed by hybridization with the 5' tagged reporter DNAprobe and a 5' phosphorylated probe. The sample is incubated with T4 DNAligase. The DNA strands with ligated probes are captured on the array byhybridization to an array, wherein non-ligated products do nothybridize. The tags are cleaved from the separated fragments, and thenthe tags are detected by the respective detection technology (e.g., massspectrometry, infrared spectrophotometry, potentiostatic amperometry orUV/visible spectrophotometry.

21. Other assays

The methods described herein may also be used to genotype oridentification of viruses or microbes. For example, F+ RNA coliphagesmay be useful candidates as indicators for enteric virus contamination.Genotyping by nucleic acid amplification and hybridization methods arereliable, rapid, simple, and inexpensive alternatives to serotyping(Kafatos et. al., Nucleic Acids Res. 7:1541, 1979). Amplificationtechniques and nucleic aid hybridization techniques have beensuccessfully used to classify a variety of microorganisms including E.coli (Feng, Mol. Cell Probes 7:151, 1993), rotavirus (Sethabutr et. al.,J. Med Virol. 37:192, 1992), hepatitis C virus (Stuyver et. al., J. GenVirol: 74:1093, 1993), and herpes simplex virus (Matsumoto et. al., J.Virol. Methods 40:119, 1992).

Genetic alterations have been described in a variety of experimentalmammalian and human neoplasms and represent the morphological basis forthe sequence of morphological alterations observed in carcinogenesis(Vogelstein et al., NEJM319:525, 1988). In recent years with the adventof molecular biology techniques, allelic losses on certain chromosomesor mutation of tumor suppressor genes as well as mutations in severaloncogenes (e.g., c-myc, c-jun, and the ras family) have been the moststudied entities. Previous work (Finkelstein et al., Arch Surg. 128:526,1993) has identified a correlation between specific types of pointmutations in the K-ras oncogene and the stage at diagnosis in colorectalcarcinoma. The results suggested that mutational analysis could provideimportant information of tumor aggressiveness, including the pattern andspread of metastasis. The prognostic value of TP53 and K-ras-2mutational analysis in stage III carconoma of the colon has morerecently been demonstrated (Pricolo et al., Am. J. Surg. 171:41, 1996).It is therefore apparent that genotyping of tumors and pre-cancerouscells, and specific mutation detection will become increasinglyimportant in the treatment of cancers in humans.

The tagged biomolecules as disclosed herein may be used to interrogate(untagged) arrays of biomolecules. Preferred arrays of biomolculescontain a solid substrate comprising a surface, where the surface is atleast partially covered with a layer of poly(ethylenimine) (PEI). ThePEI layer comprises a plurality of discrete first regions abutted andsurrounded by a contiguous second region. The first regions are definedby the presence of a biomolecule and PEI, while the second region isdefined by the presence of PEI and the substantial absence of thebiomolecule. Preferably, the substrate is a glass plate or a siliconwafer. However, the substrate may be, for example, quartz, gold,nylon-6,6, nylon or polystyrene, as well as composites thereof, asdescribed above.

The PEI coating preferably contains PEI having a molecular weightranging from 100 to 100,000. The PEI coating may be directly bonded tothe substrate using, for example, silylated PEI. Alternatively, areaction product of a bifunctional coupling agent may be disposedbetween the substrate surface and the PEI coating, where the reactionproduct is covalently bonded to both the surface and the PEI coating,and secures the PEI coating to the surface. The bifunctional couplingagent contains a first and a second reactive functional group, where thefirst reactive functional group is, for example, a tri(O--C₁ -C₅alkyl)silane, and the second reactive functional group is, for example,an epoxide, isocyanate, isothiocyanate and anhydride group. Preferredbifunctional coupling agents include2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane;3,4-epoxybutyltrimethoxysilane; 3-isocyanatopropyltriethoxysilane,3-(triethoxysilyl)-2-methylpropylsuccinic anhydride and3-(2,3-epoxypropoxy)propyltrimethoxysilane.

The array of the invention contains first, biomolecule-containingregions, where each region has an area within the range of about 1,000square microns to about 100,000 square microns. In a preferredembodiment, the first regions have areas that range from about 5,000square microns to about 25,000 square microns.

The first regions are preferably substantially circular, where thecircles have an average diameter of about 10 microns to 200 microns.Whether circular or not, the boundaries of the first regions arepreferably separated from one another (by the second region) by anaverage distance of at least about 25 microns, however by not more thanabout 1 cm (and preferably by no more than about 1,000 microns). In apreferred array, the boundaries of neighboring first regions areseparated by an average distance of about 25 microns to 100 microns,where that distance is preferably constant throughout the array, and thefirst regions are preferably positioned in a repeating geometric patternas shown in the Figures attached hereto. In a preferred repeatinggeometric pattern, all neighboring first regions are separated byapproximately the same distance (about 25 microns to about 100 microns).

In preferred arrays, there are from 10 to 50 first regions on thesubstrate. In another embodiment, there are 50 to 400 first regions on asubstrate. In yet another preferred embodiment, there are 400 to 800first regions on the substrate.

The biomolecule located in the first regions is preferably a nucleicacid polymer. A preferred nucleic acid polymer is an oligonucleotidehaving from about 15 to about 50 nucleotides. The biomolecule may beamplification reaction products having from about 50 to about 1,000nucleotides.

In each first region, the biomolecule is preferably present at anaverage concentration ranging from 10⁵ to 10⁹ biomolecules per 2,000square microns of a first region. More preferably, the averageconcentration of biomolecule ranges from 10⁷ to 10⁹ biomolecules per2,000 square microns. In the second region, the biomolecule ispreferably present at an average concentration of less than 10³biomolecules per 2,000 square microns of said second region, and morepreferably at an average concentration of less than 10² biomolecules per2,000 square microns. Most preferably, the second regions does notcontain any biomolecule.

The chemistry used to adhere the layer of PEI to the substrate depends,in substantial part, upon the chemical identity of the substrate. Theprior art provides numerous examples of suitable chemistries that mayadhere PEI to a solid support. For example, when the substrate isnylon-6,6, the PEI coating may be applied by the methods disclosed inVan Ness, J. et al. Nucleic Acids Res. 19:3345-3350, 1991 and PCTInternational Publication WO 94/00600, both of which are incorporatedherein by reference. When the solid support is glass or silicon,suitable methods of applying a layer of PEI are found in, e.g.,Wasserman, B. P. Biotechnology and Bioengineering XXII:271-287, 1980;and D'Souza, S. F. Biotechnology Letters 8:643-648, 1986.

Preferably, the PEI coating is covalently attached to the solidsubstrate. When the solid substrate is glass or silicon, the PEI coatingmay be covalently bound to the substrate using silylating chemistry. Forexample, PEI having reactive siloxy endgroups is commercially availablefrom Gelest, Inc. (Tullytown, Pa.,). Such reactive PEI may be contactedwith a glass slide or silicon wafer, and after gentle agitation, the PEIwill adhere to the substrate. Alternatively, a bifunctional silylatingreagent may be employed. According to this process, the glass or siliconsubstrate is treated with the bifunctional silylating reagent to providethe substrate with a reactive surface. PEI is then contacted with thereactive surface, and covalently binds to the surface through thebifunctional reagent.

The biomolecules being placed into the array format are originallypresent in a so-called "arraying solution". In order to placebiomolecule in discrete regions on the PEI-coated substrate, thearraying solution preferably contains a thickening agent at aconcentration of about 35 vol % to about 80 vol % based on the totalvolume of the composition, a biomolecule which is preferably anoligonucleotide at a concentration ranging from 0.001 μg/mL to 10 μg/mL,and water.

The concentration of the thickening agent is 35% V/V to 80% V/V forliquid thickening agents such as glycerol. The preferred concentrationof thickening agent in the composition depends, to some extent, on thetemperature at which the arraying is performed. The lower the arrayingtemperature, the lower the concentration of thickening agent that needsto be used. The combination of temperature and liquid thickening agentconcentration control permits arrays to be made on most types of solidsupports (e.g, glass, wafers, nylon 6/6, nylon membranes, etc.).

The presence of a thickening agent has the additional benefit ofallowing the concurrent presence of low concentrations of various othermaterials to be present in combination with the biomolecule. For example0.001% V/V to 1% V/V of detergents may be present in the arrayingsolution. This is useful because PCR buffer contains a small amount ofTween-20 or NP-40, and it is frequently desirable to array samplenucleic acids directly from a PCR vial without prior purification of theamplicons. The use of a thickening agent permits the presence of salts(for example NaCl, KCl, or MgCl₂), buffers (for example Tris), and/orchelating reagents (for example EDTA) to also be present in the arrayingsolution. The use of a thickening agent also has the additional benefitof permitting the use of cross-linking reagents and/or organic solventsto be present in the arraying solution. As commercially obtained,cross-linking reagents are commonly dissolved in organic solvent such asDMSO, DMF, NMP, methanol, ethanol and the like. Commonly used organicsolvents can be used in arraying solutions of the invention at levels of0.05% to 20% (V/V) when thickening agents are used.

In general, the thickening agents impart increased viscosity to thearraying solution. When a proper viscosity is achieved in the arrayingsolution, the first drop is the substantially the same size as, forexample, the 100th drop deposited. When an improper viscosity is used inthe arraying solution, the first drops deposited are significantlylarger than latter drops which are deposited. The desired viscosity isbetween those of pure water and pure glycerin.

The biomolecule in the array may be a nucleic acid polymer or analogthereof, such as PNA, phosphorothioates and methylphosphonates. Nucleicacid refers to both ribonucleic acid and deoxyribonucleic acid. Thebiomolecule may comprise unnatural and/or synthetic bases. Thebiomolecule may be single or double stranded nucleic acid polymer.

A preferred biomolecule is an nucleic acid polymer, which includesoligonucleotides (up to about 100 nucleotide bases) and polynucleotides(over about 100 bases). A preferred nucleic acid polymer is formed from15 to 50 nucleotide bases. Another preferred nucleic acid polymer has 50to 1,000 nucleotide bases. The nucleic acid polymer may be a PCRproduct, PCR primer, or nucleic acid duplex, to list a few examples.However, essentially any nucleic acid type can be covalently attached toa PEI-coated surface when the nucleic acid contains a primary amine, asdisclosed below. The typical concentration of nucleic acid polymer inthe arraying solution is 0.001-10 μg/mL, preferably 0.01-1 μg/mL, andmore preferably 0.05-0.5 μg/mL.

Preferred nucleic acid polymers are "amine-modified" in that they havebeen modified to contain a primary amine at the 5'-end of the nucleicacid polymer, preferably with one or more methylene (--CH₂ --) groupsdisposed between the primary amine and the nucleic acid portion of thenucleic acid polymer. Six is a preferred number of methylene groups.Amine-modified nucleic acid polymers are preferred because they can becovalently coupled to a solid support through the 5 '-amine group. PCRproducts can be arrayed using 5'-hexylamine modified PCR primers.Nucleic acid duplexes can be arrayed after the introduction of amines bynick translation using aminoallyl-dUTP (Sigma, St. Louis, Mo.). Aminescan be introduced into nucleic acids by polymerases such as terminaltransferase with amino allyl-dUTP or by ligation of shortamine-containing nucleic acid polymers onto nucleic acids by ligases.

Preferably, the nucleic acid polymer is activated prior to be contactedwith the PEI coating. This can be conveniently accomplished by combiningamine-functionalized nucleic acid polymer with a multi-functionalamine-reactive chemical such as trichlorotriazine. When the nucleic acidpolymer contains a 5'-amine group, that 5'-amine can be reacted withtrichlorotriazine, also known as cyanuric chloride (Van Ness et al.,Nucleic Acids Res. 19(2):3345-3350, 1991) Preferably, an excess ofcyanuric chloride is added to the nucleic acid polymer solution, where a10- to 1000-fold molar excess of cyanuric chloride over the number ofamines in the nucleic acid polymer in the arraying solution ispreferred. In this way, the majority of amine-terminated nucleic acidpolymers have reacted with one molecule of trichlorotriazine, so thatthe nucleic acid polymer becomes terminated with dichlorotriazine.

Preferably, the arraying solution is buffered using a common buffer suchas sodium phosphate, sodium borate, sodium carbonate, or Tris HCl. Apreferred pH range for the arraying solution is 7 to 9. with a preferredbuffer being freshly prepared sodium borate at pH 8.3 to pH 8.5. Toprepare a typical arraying solution, hexylamine-modified nucleic acidpolymer is placed in 0.2 M sodium borate, pH 8.3, at 0.1 μg/mL, to atotal volume of 50 μl. Ten μl of a 15 mg/mL solution of cyanuricchloride is then added, and the reaction is allowed to proceed for 1hour at 25 C with constant agitation. Glycerol (Gibco Brl®, GrandIsland, N.Y.) is added to a final concentration of 56%.

The biomolecular arraying solutions may be applied to the PEI coating byany of the number of techniques currently used in microfabrication. Forexample, the solutions may be placed into an ink jet print head, andejected from such a head onto the coating.

A preferred approach to delivering biomolecular solution onto the PEIcoating employs a modified spring probe. Spring probes are availablefrom several vendors including Everett Charles (Pomona, Calif.),Interconnect Devices Inc. (Kansas City, Kans.) and Test ConnectionsInc., (Upland, Calif.). In order for the commercially available springprobes as described above to satisfactorily function as liquiddeposition devices according to the present invention, approximately1/1000th to 5/1000th of an inch of metal material must be removed fromthe tip of the probe. The process must result in a flat surface which isperpendicular to the longitudinal axis of the spring probe. The removalof approximately 1/1000th to 5/1000th of an inch of material from thebottom of the tip is preferred and can be accomplished easily with avery fine grained wet stone. Specific spring probes which arecommercially available and may be modified to provide a planar tip asdescribed above include the XP54 probe manufactured by Ostby Barton (adivision of Everett Charles (Pomona, Calif.)); the SPA 25P probemanufactured by Everett Charles (Pomona, Calif.) and 43-P fluted springprobe from Test Connections Inc., (Upland, Calif.).

The arraying solutions as described above may be used directly in anarraying process. That is, the activated nucleic acid polymers need notbe purified away from unreacted cyanuric chloride prior to the printingstep. Typically the reaction which attaches the activated nucleic acidto the solid support is allowed to proceed for 1 to 20 hours at 20 to 50C. Preferably, the reaction time is 1 hour at 25 C.

The arrays as described herein are particularly useful in conductinghybridization assays, for example, using CMST labeled probes. However,in order to perform such assays, the amines on the solid support must becapped prior to conducting the hybridization step. This may beaccomplished by reacting the solid support with 0.1-2.0 M succinicanhydride. The preferred reaction conditions are 1.0 M succinicanhydride in 70% m-pyrol and 0.1 M sodium borate. The reaction typicallyis allowed to occur for 15 minutes to 4 hours with a preferred reactiontime of 30 minutes at 25 C. Residual succinic anhydride is removed witha 3× water wash.

The solid support is then incubated with a solution containing 0.1-5 Mglycine in 0.1-10.0 M sodium borate at pH 7-9. This step "caps" anydichloro-triazine which may be covalently bound to the PEI surface byconversion into monochlorotriazine. The preferred conditions are 0.2 Mglycine in 0.1 M sodium borate at pH 8.3. The solid support may then bewashed with detergent-containing solutions to remove unbound materials,for example, trace NMP. Preferably, the solid support is heated to 95 Cin 0.01 M NaCl, 0.05 M EDTA and 01 M Tris pH 8.0 for 5 minutes. Thisheating step removes non-covalently attached nucleic acid polymers, suchas PCR products. In the case where double strand nucleic acid arearrayed, this step also has the effect of converting the double strandto single strand form (denaturation).

The arrays are may be interrogated by probes (e.g., oligonucleotides,nucleic acid fragments, PCR products, etc.) which may be tagged with,for example CMST tags as described herein, radioisotopes, fluorophoresor biotin. The methods for biotinylating nucleic acids are well known inthe art and are adequately described by Pierce (Avidin-Biotin Chemistry:A Handbook, Pierce Chemical Company, 1992, Rockford Ill.). Probes aregenerally used at 0.1 ng/mL to 10/μg/mL in standard hybridizationsolutions that include GuSCN, GuHCl, formamide, etc. (see Van Ness andChen, Nucleic Acids Res., 19:5143-5151, 1991).

To detect the hybridization event (i.e., the presence of the biotin),the solid support is incubated with streptavidin/horseradish peroxidaseconjugate. Such enzyme conjugates are commercially available from, forexample, Vector Laboratories (Burlingham, Calif.). The streptavidinbinds with high affinity to the biotin molecule bringing the horseradishperoxidase into proximity to the hybridized probe. Unboundstreptavidin/horseradish peroxidase conjugate is washed away in a simplewashing step. The presence of horseradish peroxidase enzyme is thendetected using a precipitating substrate in the presence of peroxide andthe appropriate buffers.

A blue enzyme product deposited on a reflective surface such as a waferhas a many-fold lower level of detection (LLD) compared to that expectedfor a colorimetric substrate. Furthermore, the LLD is vastly differentfor different colored enzyme products. For example, the LLD for4-methoxynapthol (which produces a precipitated blue product) per 50 μMdiameter spot is approximately 1000 molecules, whereas a redprecipitated substrate gives an LLD about 1000-fold higher at 1,000,000molecules per 50 μM diameter spot. The LLD is determined byinterrogating the surface with a microscope (such as the Axiotechmicroscope commercially available from Zeiss) equipped with a visiblelight source and a CCD camera (Princeton Instruments, Princeton, N.J.).An image of approximately 10,000 μM×10,000 μM can be scanned at onetime.

In order to use the blue colorimetric detection scheme, the surface mustbe very clean after the enzymatic reaction and the wafer or slide mustbe scanned in a dry state. In addition, the enzymatic reaction must bestopped prior to saturation of the reference spots. For horseradishperoxidase this is approximately 2-5 minutes.

It is also possible to use chemiluminescent substrates for alkalinephosphatase or horesradish peroxidase (HRP), or fluoroescence substratesfor HRP or alkaline phosphatase. Examples include the dioxetanesubstrates for alkaline phosphatase available from Perkin Elmer orAttophos HRP substrate from JBL Scientific (San Luis Obispo, Calif.).

The following examples are offered by way of illustration, and not byway of limitation.

Unless otherwise stated, chemicals as used in the examples may beobtained from Aldrich Chemical Company, Milwaukee, Wis. The followingabbreviations, with the indicated meanings, are used herein:

ANP=3-(Fmoc-amino)-3-(2-nitrophenyl)propionic acid

NBA=4-(Fmoc-aminomethyl)-3-nitrobenzoic acid

HATU=O-7-azabenzotriazol-1-yl-N,N,N',N'-tetramethyluroniumhexafluoro-phosphate

DIEA=diisopropylethylamine

MCT=monochlorotriazine

NMM=4-methylmorpholine

NMP=N-methylpyrrolidone

ACT357=ACT357 peptide synthesizer from Advanced ChemTech, Inc.,Louisville, Ky.

ACT=Advanced ChemTech, Inc., Louisville, Ky.

NovaBiochem=CalBiochem-NovaBiochem International, San Diego, Calif.

TFA=Trifluoroacetic acid

Tfa=Trifluoroacetyl

iNIP=N-Methylisonipecotic acid

Tfp=Tetrafluorophenyl

DIAEA=2-(Diisopropylamino)ethylamine

MCT=monochlorotriazene

5'-AH-ODN=5'-aminohexyl-tailed oligodeoxynucleotide

EXAMPLES Example 1 Preparation of Acid Labile Linkers For Use inCleavable-Tag Sequencing

A. Synthesis of Pentafluorophenyl Esters of Chemically Cleavable MassSpectroscopy Tags, to Liberate Tags with Carboxyl Amide Termini

FIG. 1 shows the reaction scheme.

Step A

TentaGel S AC resin (compound II; available from ACT; 1 eq.) issuspended with DMF in the collection vessel of the ACT357 peptidesynthesizer (ACT). Compound I (3 eq.), HATU (3 eq.) and DIEA (7.5 eq.)in DMF are added and the collection vessel shaken for 1 hr. The solventis removed and the resin washed with NMP (2×), MeOH (2×), and DMF (2×).The coupling of 1 to the resin and the wash steps are repeated, to givecompound III.

Step B

The resin (compound III) is mixed with 25% piperidine in DMF and shakenfor min. The resin is filtered, then mixed with 25% piperidine in DMFand shaken for 10 min. The solvent is removed, the resin washed with NMP(2×), MeOH (2×), and DMF (2×), and used directly in step C.

Step C

The deprotected resin from step B is suspended in DMF and to it is addedan FMOC-protected amino acid, containing amine functionality in its sidechain (compound IV, e.g. alpha-N-FMOC-3-(3-pyridyl)-alanine, availablefrom Synthetech, Albany, Oreg.; 3 eq.), HATU (3 eq.), and DIEA (7.5 eq.)in DMF. The vessel is shaken for 1 hr. The solvent is removed and theresin washed with NMP (2×), MeOH (2×), and DMF (2×). The coupling of IVto the resin and the wash steps are repeated, to give compound V.

Step D

The resin (compound V) is treated with piperidine as described in step Bto remove the FMOC group. The deprotected resin is then divided equallyby the ACT357 from the collection vessel into 16 reaction vessels.

Step E

The 16 aliquots of deprotected resin from step D are suspended in DMF.To each reaction vessel is added the appropriate carboxylic acid VI₁₋₁₆(R₁₋₁₆ CO₂ H; 3 eq.), HATU (3 eq.), and DIEA (7.5 eq.) in DMF. Thevessels are shaken for 1 hr. The solvent is removed and the aliquots ofresin washed with NMP (2×), MeOH (2×), and DMF (2×). The coupling ofVI₁₋₁₆ to the aliquots of resin and the wash steps are repeated, to givecompounds VII₁₋₁₆.

Step F

The aliquots of resin (compounds VII₁₋₁₆) are washed with CH₂ Cl₂ (3×).To each of the reaction vessels is added 1% TFA in CH₂ Cl₂ and thevessels shaken for 30 min. The solvent is filtered from the reactionvessels into individual tubes. The aliquots of resin are washed with CH₂Cl₂ (2×) and MeOH (2×) and the filtrates combined into the individualtubes. The individual tubes are evaporated in vacuo, providing compoundsVIII₁₋₁₆.

Step G

Each of the free carboxylic acids VIII₁₋₁₆ is dissolved in DMF. To eachsolution is added pyridine (1.05 eq.), followed by pentafluorophenyltrifluoroacetate (1.1 eq.). The mixtures are stirred for 45 min. at roomtemperature. The solutions are diluted with EtOAc, washed with 1 M aq.citric acid (3×) and 5% aq. NaHCO₃ (3×), dried over Na₂ SO₄, filtered,and evaporated in vacuo, providing compounds IX₁₋₁₆.

B. Synthesis of Pentafluorophenyl Esters of Chemically Cleavable MassSpectroscopy Tags, to Liberate Tags with Carboxyl Acid Termini

FIG. 2 shows the reaction scheme.

Step A

4-(Hydroxymethyl)phenoxybutyric acid (compound I; 1 eq.) is combinedwith DIEA (2.1 eq.) and allyl bromide (2.1 eq.) in CHCl₃ and heated toreflux for 2 hr. The mixture is diluted with EtOAc, washed with 1 N HCl(2×), pH 9.5 carbonate buffer (2×), and brine (1×), dried over Na₂ SO₄,and evaporated in vacuo to give the allyl ester of compound I.

Step B

The allyl ester of compound I from step A (1.75 eq.) is combined in CH₂Cl₂ with an FMOC-protected amino acid containing amine functionality inits side chain (compound II, e.g. alpha-N-FMOC-3-(3-pyridyl)-alanine,available from Synthetech, Albany, Oreg.; 1 eq.), N-methylmorpholine(2.5 eq.), and HATU (1.1 eq.), and stirred at room temperature for 4 hr.The mixture is diluted with CH₂ Cl₂, washed with 1 M aq. citric acid(2×), water (1×), and 5% aq. NaHCO₃ (2×), dried over Na₂ SO₄, andevaporated in vacuo. Compound III is isolated by flash chromatography(CH₂ Cl₂ →EtOAc).

Step C

Compound III is dissolved in CH₂ Cl₂, Pd(PPh₃)₄ (0.07 eq.) andN-methylaniline (2 eq.) are added, and the mixture stirred at roomtemperature for 4 hr. The mixture is diluted with CH₂ Cl₂, washed with 1M aq. citric acid (2×) and water (1×), dried over Na₂ SO₄, andevaporated in vacuo. Compound IV is isolated by flash chromatography(CH₂ Cl₂ →EtOAc+HOAc).

Step D

TentaGel S AC resin (compound V; 1 eq.) is suspended with DMF in thecollection vessel of the ACT357 peptide synthesizer (Advanced ChemTechInc. (ACT), Louisville, Ky.). Compound IV (3 eq.), HATU (3 eq.) and DIEA(7.5 eq.) in DMF are added and the collection vessel shaken for 1 hr.The solvent is removed and the resin washed with NMP (2×), MeOH (2×),and DMF (2×). The coupling of IV to the resin and the wash steps arerepeated, to give compound VI.

Step E

The resin (compound VI) is mixed with 25% piperidine in DMF and shakenfor 5 min. The resin is filtered, then mixed with 25% piperidine in DMFand shaken for 10 min. The solvent is removed and the resin washed withNMP (2×), MeOH (2×), and DMF (2×). The deprotected resin is then dividedequally by the ACT357 from the collection vessel into 16 reactionvessels.

Step F

The 16 aliquots of deprotected resin from step E are suspended in DMF.To each reaction vessel is added the appropriate carboxylic acid VII₁₋₁₆(R₁₋₁₆ CO₂ H; 3 eq.), HATU (3 eq.), and DIEA (7.5 eq.) in DMF. Thevessels are shaken for 1 hr. The solvent is removed and the aliquots ofresin washed with NMP (2×), MeOH (2×), and DMF (2×). The coupling ofVII₁₋₁₆ to the aliquots of resin and the wash steps are repeated, togive compounds VIII₁₋₁₆.

Step G

The aliquots of resin (compounds VIII₁₋₁₆) are washed with CH₂ Cl₂ (3×).To each of the reaction vessels is added 1% TFA in CH₂ Cl₂ and thevessels shaken for 30 min. The solvent is filtered from the reactionvessels into individual tubes. The aliquots of resin are washed with CH₂Cl₂ (2×) and MeOH (2×) and the filtrates combined into the individualtubes. The individual tubes are evaporated in vacuo, providing compoundsIX₁₋₁₆.

Step H. Each of the free carboxylic acids IX₁₋₁₆ is dissolved in DMF. Toeach solution is added pyridine (1.05 eq.), followed bypentafluorophenyl trifluoroacetate (1.1 eq.). The mixtures are stirredfor 45 min. at room temperature. The solutions are diluted with EtOAc,washed with 1 M aq. citric acid (3×) and 5% aq. NaHCO₃ (3×), dried overNa₂ SO₄, filtered, and evaporated in vacuo, providing compounds X₁₋₁₆.

Example 2 Demonstration of Photolytic Cleavage of T-L-X

A T-L-X compound as prepared in Example 11 was irradiated with near-UVlight for 7 min at room temperature. A Rayonett fluorescence UV lamp(Southern New England Ultraviolet Co., Middletown, Conn.) with anemission peak at 350 nm is used as a source of UV light. The lamp isplaced at a 15-cm distance from the Petri dishes with samples. SDS gelelectrophoresis shows that >85% of the conjugate is cleaved under theseconditions.

Example 3 Preparation of Fluorescent Labeled Primers and Demonstrationof Cleavage of Fluorophore

Synthesis and Purification of Oligonucleotides

The oligonucleotides (ODNs) are prepared on automated DNA synthesizersusing the standard phosphoramidite chemistry supplied by the vendor, orthe H-phosphonate chemistry (Glenn Research Sterling, Va.).Appropriately blocked dA, dG, dC, and T phosphoramidites arecommercially available in these forms, and synthetic nucleosides mayreadily be converted to the appropriate form. The oligonucleotides areprepared using the standard phosphoramidite supplied by the vendor, orthe H-phosphonate chemistry. Oligonucleotides are purified byadaptations of standard methods. Oligonucleotides with 5'-trityl groupsare chromatographed on HPLC using a 12 micrometer, 300# Rainin(Emeryville, Calif.) Dynamax C-8 4.2×250 mm reverse phase column using agradient of 15% to 55% MeCN in 0.1 N Et₃ NH⁺ OAc⁺, pH 7.0, over 20 min.When detritylation is performed, the oligonucleotides are furtherpurified by gel exclusion chromatography. Analytical checks for thequality of the oligonucleotides are conducted with a PRP-column(Alltech, Deerfield, Ill.) at alkaline pH and by PAGE.

Preparation of 2,4,6-trichlorotriazine derived oligonucleotides: 10 to1000 μg of 5'-terminal amine linked oligonucleotide are reacted with anexcess recrystallized cyanuric chloride in 10% n-methyl-pyrrolidone inalkaline (pH 8.3 to 8.5 preferably) buffer at 19° C. to 25° C. for 30 to120 minutes. The final reaction conditions consist of 0.15 M sodiumborate at pH 8.3, 2 mg/ml recrystallized cyanuric chloride and 500 ug/mlrespective oligonucleotide. The unreacted cyanuric chloride is removedby size exclusion chromatography on a G-50 Sephadex (Pharmacia,Piscataway, N.J.) column.

The activated purified oligonucleotide is then reacted with a 100-foldmolar excess of cystamine in 0.15 M sodium borate at pH 8.3 for 1 hourat room temperature. The unreacted cystamine is removed by sizeexclusion chromatography on a G-50 Sephadex column. The derived ODNs arethen reacted with amine-reactive fluorochromes. The derived ODNpreparation is divided into 3 portions and each portion is reacted witheither (a) 20-fold molar excess of Texas Red sulfonyl chloride(Molecular Probes, Eugene, Oreg.), with (b) 20-fold molar excess ofLissamine sulfonyl chloride (Molecular Probes, Eugene, Oreg.), (c)20-fold molar excess of fluorescein isothiocyanate. The final reactionconditions consist of 0.15 M sodium borate at pH 8.3 for 1 hour at roomtemperature. The unreacted fluorochromes are removed by size exclusionchromatography on a G-50 Sephadex column.

To cleave the fluorochrome from the oligonucleotide, the ODNs areadjusted to 1×10⁻⁵ molar and then dilutions are made (12, 3-folddilutions) in TE (TE is 0.01 M Tris, pH 7.0, 5 mM EDTA). To 100 μlvolumes of ODNs 25 μl of 0.01 M dithiothreitol (DTT) is added. To anidentical set of controls no DDT is added. The mixture is incubated for15 minutes at room temperature. Fluorescence is measured in a blackmicrotiter plate. The solution is removed from the incubation tubes (150microliters) and placed in a black microtiter plate (DynatekLaboratories, Chantilly, Va.). The plates are then read directly using aFluoroskan II fluorometer (Flow Laboratories, McLean, Va.) using anexcitation wavelength of 495 nm and monitoring emission at 520 nm forfluorescein, using an excitation wavelength of 591 nm and monitoringemission at 612 nm for Texas Red, and using an excitation wavelength of570 nm and monitoring emission at 590 nm for lissamine.

    ______________________________________                                        Moles of   RFU           RFU     RFU                                            Fluorochrome non-cleaved cleaved free                                       ______________________________________                                        1.0 × 10.sup.5 M                                                                   6.4           1200    1345                                           3.3 × 10.sup.6 M 2.4  451 456                                           1.1 × 10.sup.6 M 0.9  135 130                                           3.7 × 10.sup.7 M 0.3  44 48                                             1.2 × 10.sup.7 M 0.12 15.3 16.0                                         4.1 × 10.sup.7 M 0.14 4.9 5.1                                           1.4 × 10.sup.8 M 0.13 2.5 2.8                                           4.5 × 10.sup.9 M 0.12 0.8 0.9                                         ______________________________________                                    

The data indicate that there is about a 200-fold increase in relativefluorescence when the fluorochrome is cleaved from the ODN.

Example 4 Preparation of Tagged M13 Sequence Primers and Demonstrationof Cleavage of Tags

Preparation of 2,4,6-trichlorotriazine derived oligonucleotides: 1000 μgof 5'-terminal amine linked oligonucleotide(5'-hexylamine-TGTAAAACGACGGCCAGT-3") (Seq. ID No. 1) are reacted withan excess recrystallized cyanuric chloride in 10% n-methyl-pyrrolidonealkaline (pH 8.3 to 8.5 preferably) buffer at 19 to 25- C for 30 to 120minutes. The final reaction conditions consist of 0.15 M sodium borateat pH 8.3, 2 mg/ml recrystallized cyanuric chloride and 500 ug/mlrespective oligonucleotide. The unreacted cyanuric chloride is removedby size exclusion chromatography on a G-50 Sephadex column.

The activated purified oligonucleotide is then reacted with a 100-foldmolar excess of cystamine in 0.15 M sodium borate at pH 8.3 for 1 hourat room temperature. The unreacted cystamine is removed by sizeexclusion chromatography on a G-50 Sephadex column. The derived ODNs arethen reacted with a variety of amides.

The derived ODN preparation is divided into 12 portions and each portionis reacted (25 molar excess) with the pentafluorophenyl-esters ofeither: (1) 4-methoxybenzoic acid, (2) 4-fluorobenzoic acid, (3) toluicacid, (4) benzoic acid, (5) indole-3-acetic acid, (6)2,6-difluorobenzoic acid, (7) nicotinic acid N-oxide, (8) 2-nitrobenzoicacid, (9) 5-acetylsalicylic acid, (10) 4-ethoxybenzoic acid, (11)cinnamic acid, (12) 3-aminonicotinic acid. The reaction is for 2 hoursat 37° C. in 0.2 M NaBorate pH 8.3. The derived ODNs are purified by gelexclusion chromatography on G-50 Sephadex.

To cleave the tag from the oligonucleotide, the ODNs are adjusted to1×10⁻⁵ molar and then dilutions are made (12, 3-fold dilutions) in TE(TE is 0.01 M Tris, pH 7.0, 5 mM EDTA) with 50% EtOH (V/V). To 100 μlvolumes of ODNs 25 μl of 0.01 M dithiothreitol (DTT) is added. To anidentical set of controls no DDT is added. Incubation is for 30 minutesat room temperature. NaCl is then added to 0.1 M and 2 volumes of EtOHis added to precipitate the ODNs. The ODNs are removed from solution bycentrifugation at 14,000×G at 4° C. for 15 minutes. The supernatants arereserved, dried to completeness. The pellet is then dissolved in 25 μlMeOH. The pellet is then tested by mass spectrometry for the presence oftags.

The mass spectrometer used in this work is an external ion sourceFourier-transform mass spectrometer (FTMS). Samples prepared for MALDIanalysis are deposited on the tip of a direct probe and inserted intothe ion source. When the sample is irradiated with a laser pulse, ionsare extracted from the source and passed into a long quadrupole ionguide that focuses and transports them to an FTMS analyzer cell locatedinside the bore of a superconducting magnet.

The spectra yield the following information. Peaks varying in intensityfrom 25 to 100 relative intensity units at the following molecularweights: (1) 212.1 amu indicating 4-methoxybenzoic acid derivative, (2)200.1 indicating 4-fluorobenzoic acid derivative, (3) 196.1 amuindicating toluic acid derivative, (4) 182.1 amu indicating benzoic acidderivative, (5) 235.2 amu indicating indole-3-acetic acid derivative,(6) 218.1 amu indicating 2,6-difluorobenzoic derivative, (7) 199.1 amuindicating nicotinic acid N-oxide derivative, (8) 227.1 amu indicating2-nitrobenzamide, (9) 179.18 amu indicating 5-acetylsalicylic acidderivative, (10) 226.1 amu indicating 4-ethoxybenzoic acid derivative,(11) 209.1 amu indicating cinnamic acid derivative, (12) 198.1 amuindicating 3-aminonicotinic acid derivative.

The results indicate that the tags are cleaved from the primers and aredetectable by mass spectrometry.

Example 5 Preparation of a Set of Compounds of the Formula R₁₋₃₆-Lys(ε-iNIP)-ANP-Tfp

FIG. 3 illustrates the parallel synthesis of a set of 36 T-L-X compounds(X=L_(h)), where L_(h) is an activated ester (specifically,tetrafluorophenyl ester), L² is an ortho-nitrobenzylamine group with L³being a methylene group that links L_(h) and L², T has a modularstructure wherein the carboxylic acid group of lysine has been joined tothe nitrogen atom of the L² benzylamine group to form an amide bond, anda variable weight component R₁₋₃₆, (where these R groups correspond toT² as defined herein, and may be introduced via any of the specificcarboxylic acids listed herein) is bonded through the α-amino group ofthe lysine, while a mass spec sensitivity enhancer group (introduced viaN-methylisonipecotic acid) is bonded through the ε-amino group of thelysine.

Referring to FIG. 3:

Step A

NovaSyn HMP Resin (available from NovaBiochem; 1 eq.) is suspended withDMF in the collection vessel of the ACT357. Compound I (ANP availablefrom ACT; 3 eq.), HATU (3 eq.) and NMM (7.5 eq.) in DMF are added andthe collection vessel shaken for 1 hr. The solvent is removed and theresin washed with NMP (2×), MeOH (2×), and DMF (2×). The coupling of 1to the resin and the wash steps are repeated, to give compound II.

Step B

The resin (compound II) is mixed with 25% piperidine in DMF and shakenfor 5 min. The resin is filtered, then mixed with 25% piperidine in DMFand shaken for 10 min. The solvent is removed, the resin washed with NMP(2×), MeOH (2×), and DMF (2×), and used directly in step C.

Step C

The deprotected resin from step B is suspended in DMF and to it is addedan FMOC-protected amino acid, containing a protected amine functionalityin its side chain (Fmoc-Lysine(Aloc)-OH, available from PerSeptiveBiosystems; 3 eq.), HATU (3 eq.), and NMM (7.5 eq.) in DMF. The vesselis shaken for 1 hr. The solvent is removed and the resin washed with NMP(2×), MeOH (2×), and DMF (2×). The coupling of Fmoc-Lys(Aloc)-OH to theresin and the wash steps are repeated, to give compound IV.

Step D

The resin (compound IV) is washed with CH₂ Cl₂ (2×), and then suspendedin a solution of (PPh₃)₄ Pd (0) (0.3 eq.) and PhSiH₃ (10 eq.) in CH₂Cl₂. The mixture is shaken for 1 hr. The solvent is removed and theresin is washed with CH₂ Cl₂ (2×). The palladium step is repeated. Thesolvent is removed and the resin is washed with CH₂ Cl₂ (2×),N,N-diisopropylethylammonium diethyldithiocarbamate in DMF (2×), DMF(2×) to give compound V.

Step E

The deprotected resin from step D is coupled with N-methylisonipecoticacid as described in step C to give compound VI.

Step F

The Fmoc protected resin VI is divided equally by the ACT357 from thecollection vessel into 36 reaction vessels to give compounds VI₁₋₃₆.

Step G

The resin (compounds VI₁₋₃₆) is treated with piperidine as described instep B to remove the FMOC group.

Step H

The 36 aliquots of deprotected resin from step GJ are suspended in DMF.To each reaction vessel is added the appropriate carboxylic acid (R₁₋₃₆CO₂ H; 3 eq.), HATU (3 eq.), and NMM (7.5 eq.) in DMF. The vessels areshaken for 1 hr. The solvent is removed and the aliquots of resin washedwith NMP (2×), MeOH (2×), and DMF (2×). The coupling of R₁₋₃₆ CO₂ H tothe aliquots of resin and the wash steps are repeated, to give compoundsVIII₁₋₃₆.

Step I. The aliquots of resin (compounds VIII₁₋₃₆) are washed with CH₂Cl₂ (3×). To each of the reaction vessels is added 90:5:5 TFA:H20:CH₂Cl₂ and the vessels shaken for 120 min. The solvent is filtered from thereaction vessels into individual tubes. The aliquots of resin are washedwith CH₂ Cl₂ (2×) and MeOH (2×) and the filtrates combined into theindividual tubes. The individual tubes are evaporated in vacuo,providing compounds IX₁₋₃₆.

Step J

Each of the free carboxylic acids IX₁₋₃₆ is dissolved in DMF. To eachsolution is added pyridine (1.05 eq.), followed by tetrafluorophenyltrifluoroacetate (1.1 eq.). The mixtures are stirred for 45 min. at roomtemperature. The solutions are diluted with EtOAc, washed with 5% aq.NaHCO₃ (3×), dried over Na₂ SO₄, filtered, and evaporated in vacuo,providing compounds X₁₋₃₆.

Example 6 Preparation of a Set of Compounds of the Formula R₁₋₃₆-Lys(F-iNIP)-NBA-Tfp

FIG. 4 illustrates the parallel synthesis of a set of 36 T-L-X compounds(X=L_(h)), where L_(h) is an activated ester (specifically,tetrafluorophenyl ester), L² is an ortho-nitrobenzylamine group with L³being a direct bond between L_(h) and L², where L_(h) is joined directlyto the aromatic ring of the L² group, T has a modular structure whereinthe carboxylic acid group of lysine has been joined to the nitrogen atomof the L² benzylamine group to form an amide bond, and a variable weightcomponent R₁₋₃₆, (where these R groups correspond to T² as definedherein, and may be introduced via any of the specific carboxylic acidslisted herein) is bonded through the α-amino group of the lysine, whilea mass spec enhancer group (introduced via N-methylisonipecotic acid) isbonded through the ε-amino group of the lysine.

Referring to FIG. 4

Step A

NovaSyn HMP Resin is coupled with compound I (NBA prepared according tothe procedure of Brown et al., Molecular Diversity, 1, 4 (1995))according to the procedure described in step A of Example 5, to givecompound II.

Steps B-J

The resin (compound II) is treated as described in steps B-J of Example5 to give compounds X₁₋₃₆.

Example 7 Preparation of a Set of Compounds of the Formula iNIP-Lys(ε-R₁₋₃₆)-ANP-Tfp

FIG. 5 illustrates the parallel synthesis of a set of 36 T-L-X compounds(X=L_(h)), where L_(h) is an activated ester (specifically,tetrafluorophenyl ester), L² is an ortho-nitrobenzylamine group with L³being a methylene group that links Lb and L², T has a modular structurewherein the carboxylic acid group of lysine has been joined to thenitrogen atom of the L² benzylamine group to form an amide bond, and avariable weight component R₁₋₃₆, (where these R groups correspond to T²as defined herein, and may be introduced via any of the specificcarboxylic acids listed herein) is bonded through the ε-amino group ofthe lysine, while a mass spec sensitivity enhancer group (introduced viaN-methylisonipecotic acid) is bonded through the α-amino group of thelysine.

Referring to FIG. 5:

Steps A-C

Same as in Example 5.

Step D

The resin (compound IV) is treated with piperidine as described in stepB of Example 5 to remove the FMOC group.

Step E

The deprotected α-amine on the resin in step D is coupled withN-methylisonipecotic acid as described in step C of Example 5 to givecompound V.

Step F

Same as in Example 5.

Step G

The resin (compounds VI₁₋₃₆) are treated with palladium as described instep D of Example 5 to remove the Aloc group.

Steps H-J

The compounds X₁₋₃₆ are prepared in the same manner as in Example 5.

Example 8 Preparation of a Set of Compounds of the Formula R₁₋₃₆-GLU(γ-DIAEA)-ANP-Tfp

FIG. 6 illustrates the parallel synthesis of a set of 36 T-L-X compounds(X=L_(h)), where L_(h) is an activated ester (specifically,tetrafluorophenyl ester), L² is an ortho-nitrobenzylamine group with L³being a methylene group that links L_(h) and L², T has a modularstructure wherein the α-carboxylic acid group of glutamatic acid hasbeen joined to the nitrogen atom of the L² benzylamine group to form anamide bond, and a variable weight component R₁₋₃₆, (where these R groupscorrespond to T² as defined herein, and may be introduced via any of thespecific carboxylic acids listed herein) is bonded through the aα-aminogroup of the glutamic acid, while a mass spec sensitivity enhancer group(introduced via 2-(diisopropylamino)ethylamine) is bonded through theγ-carboxylic acid of the glutamic acid.

Referring to FIG. 6:

Steps A-B

Same as in Example 5.

Step C

The deprotected resin (compound III) is coupled to Fmoc-Glu-(OAl)-OHusing the coupling method described in step C of Example 5 to givecompound IV.

Step D

The allyl ester on the resin (compound IV) is washed with CH₂ Cl₂ (2×)and mixed with a solution of (PPh₃)₄ Pd (0) (0.3 eq.) andN-methylaniline (3 eq.) in CH₂ Cl₂. The mixture is shaken for 1 hr. Thesolvent is removed and the resin is washed with CH₂ Cl₂ (2×). Thepalladium step is repeated. The solvent is removed and the resin iswashed with CH₂ Cl₂ (2×), N,N-diisopropylethylammoniumdiethyldithiocarbamate in DMF (2×), DMF (2×) to give compound V.

Step E

The deprotected resin from step D is suspended in DMF and activated bymixing HATU (3 eq.), and NMM (7.5 eq.). The vessels are shaken for 15minutes. The solvent is removed and the resin washed with NMP (1×). Theresin is mixed with 2-(diisopropylamino)ethylamine (3 eq.) and NMM (7.5eq.). The vessels are shaken for 1 hour. The coupling of2-(diisopropylamino)ethylamine to the resin and the wash steps arerepeated, to give compound VI.

Steps F-J

Same as in Example 5.

Example 9 Preparation of a Set of Compounds of the Formula R₁₋₃₆-Lys(ε-iNIP)-ANP-Lys(ε-NH₂)-NH₂

FIG. 7 illustrates the parallel synthesis of a set of 36 T-L-X compounds(X=L_(h)), where L_(h) is an amine (specifically, the ε-amino group of alysine-derived moiety), L² is an ortho-nitrobenzylamine group with L³being a carboxamido-substituted alkyleneaminoacylalkylene group thatlinks L_(h) and L², T has a modular structure wherein the carboxylicacid group of lysine has been joined to the nitrogen atom of the L²benzylamine group to form an amide bond, and a variable weight componentR₁₋₃₆, (where these R groups correspond to T² as defined herein, and maybe introduced via any of the specific carboxylic acids listed herein) isbonded through the α-amino group of the lysine, while a mass specsensitivity enhancer group (introduced via N-methylisonipecotic acid) isbonded through the ε-amino group of the lysine.

Referring to FIG. 7:

Step A

Fmoc-Lys(Boc)-SRAM Resin (available from ACT; compound I) is mixed with25% piperidine in DMF and shaken for 5 min. The resin is filtered, thenmixed with 25% piperidine in DMF and shaken for 10 min. The solvent isremoved, the resin washed with NMP (2×), MeOH (2×), and DMF (2×), andused directly in step B.

Step B

The resin (compound II), ANP (available from ACT; 3 eq.), HATU (3 eq.)and NMM (7.5 eq.) in DMF are added and the collection vessel shaken for1 hr. The solvent is removed and the resin washed with NMP (2×), MeOH(2×), and DMF (2×). The coupling of 1 to the resin and the wash stepsare repeated, to give compound III.

Steps C-J

The resin (compound III) is treated as in steps B-I in Example 5 to givecompounds X₁₋₃₆.

Example 10 Preparation of a Set of Compounds of the Formula R₁₋₃₆-Lys(ε-Tfa)-Lys(ε-iINP)-ANP-Tfp

FIG. 8 illustrates the parallel synthesis of a set of 36 T-L-X compounds(X=L_(h)), where L_(h) is an activated ester (specifically,tetrafluorophenyl ester), L² is an ortho-nitrobenzylamine group with L³being a methylene group that links L_(h) and L², T has a modularstructure wherein the carboxylic acid group of a firsi lysine has beenjoined to the nitrogen atom of the L² benzylamine group to form an amidebond, a mass spec sensitivity enhancer group (introduced viaN-methylisonipecotic acid) is bonded through the ε-amino group of thefirst lysine, a second lysine molecle has been joined to the firstlysine through the α-amino group of the first lysine, a molecular weightadjuster group (having a trifluoroacetyl structure) is bonded throughthe ε-amino group of the second lysine, and a variable weight componentR₁₋₃₆, (where these R groups correspond to T² as defined herein, and maybe introduced via any of the specific carboxylic acids listed herein) isbonded through the α-amino group of the second lysine.

Referring to FIG. 8:

Steps A-E

These steps are identical to steps A-E in Example 5.

Step F

The resin (compound VI) is treated with piperidine as described in stepB in Example 5 to remove the FMOC group.

Step G

The deprotected resin (compound VII) is coupled to Fmoc-Lys(Tfa)-OHusing the coupling method described in step C of Example 5 to givecompound VIII.

Steps H-K

The resin (compound VIII) is treated as in steps F-J in Example 5 togive compounds XI₁₋₃₆.

Example 11 Preparation of a Set of Compounds of the Formula R₁₋₃₆-Lys(ε-iNIP)-ANP-5'-AH-ODN

FIG. 9 illustrates the parallel synthesis of a set of 36 T-L-X compounds(X=MOI, where MOI is a nucleic acid fragment, ODN) derived from theesters of Example 5 (the same procedure could be used with other T-L-Xcompounds wherein X is an activated ester). The MOI is conjugated to T-Lthrough the 5' end of the MOI, via a phosphodiester-alkyleneamine group.

Referring to FIG. 9:

Step A

Compounds XII₁₋₃₆ are prepared according to a modified biotinylationprocedure in Van Ness et al., Nucleic Acids Res., 19, 3345 (1991). To asolution of one of the 5'-aminohexyl oligonucleotides (compounds XI₁₋₃₆,1 mg) in 200 mM sodium borate (pH 8.3, 250 mL) is added one of theTetrafluorophenyl esters (compounds X₁₋₃₆ from Example 5, 100-fold molarexcess in 250 mL of NMP). The reaction is incubated overnight at ambienttemperature. The unreacted and hydrolyzed tetrafluorophenyl esters areremoved from the compounds XII₁₋₃₆ by Sephadex G-50 chromatography.

Example 12 Preparation of a Set of Compounds if the Formula R₁₋₃₆-Lys(ε-iNIP)-ANP-Lys(ε-(MCT-5,-AH-ODN))-NH₂

FIG. 10 illustrates the parallel synthesis of a set of 36 T-L-Xcompounds (X=MOI, where MOI is a nucleic acid fragment, ODN) derivedfrom the amines of Example 9 (the same procedure could be used withother T-L-X compounds wherein X is an amine). The MOI is conjugated toT-L through the 5' end of the MOI, via a phosphodiester--alkyleneaminegroup.

Referring to FIG. 10:

Step A

The 5'-[6-(4,6-dichloro-1,3,5-triazin-2-ylamino)hexyl]oligonucleotidesXII₁₋₃₆ are prepared as described in Van Ness et al., Nucleic AcidsRes., 19, 3345 (1991).

Step B

To a solution of one of the5'-[6-(4,6-dichloro-1,3,5-triazin-2-ylamino)hexyl]oligonucleotides(compounds XII₁₋₃₆ ) at a concentration of 1 mg/ml in 100 mM sodiumborate (pH 8.3) was added a 100-fold molar excess of a primary amineselected from R₁₋₃₆ -Lys(e-iNIP)-ANP-Lys(e-NH₂)--NH₂ (compounds X₁₋₃₆from Example 11). The solution is mixed overnight at ambienttemperature. The unreacted amine is removed by ultrafiltration through a3000 MW cutoff membrane (Amicon, Beverly, Mass.) using H₂ O as the washsolution (3×). The compounds XIII₁₋₃₆ are isolated by reduction of thevolume to 100 mL.

Example 13 Demonstration of the Simultaneous Detection of Multiple Tagsby Mass Spectrometry

This example provides a description of the ability to simultaneouslydetect multiple compounds (tags) by mass spectrometry. In thisparticular example, 31 compounds are mixed with a matrix, deposited anddried on to a solid support and then desorbed with a laser. Theresultant ions are then introduced in a mass spectrometer.

The following compounds (purchased from Aldrich, Milwaukee, Wis.) aremixed together on an equal molar basis to a final concentration of 0.002M (on a per compound) basis: benzamide (121.14), nicotinamide (122.13),pyrazinamide (123.12), 3-amino-4-pyrazolecarboxylic acid (127.10),2-thiophenecarboxamide (127.17), 4-aminobenzamide (135.15), tolumide(135.17), 6-methylnicotinamide (136.15), 3-aminonicotinamide (137.14),nicotinamide N-oxide (138.12), 3-hydropicolinamide (138.13),4-fluorobenzamide (139.13), cinnamamide (147.18), 4-methoxybenzamide(151.17), 2,6-difluorbenzamide (157.12),4-amino-5-imidazole-carboxyamide (162.58), 3,4-pyridine-dicarboxyamide(165.16), 4-ethoxybenzamide (165.19), 2,3-pyrazinedicarboxamide(166.14), 2-nitrobenzamide (166.14), 3-fluoro-4-methoxybenzoic acid(170.4), indole-3-acetamide (174.2), 5-acetylsalicylamide (179.18),3,5-dimethoxybenzamide (181.19), 1-naphthaleneacetamide (185.23),8-chloro-3,5-diamino-2-pyrazinecarboxyamide (187.59),4-trifluoromethyl-benzamide (189.00),5-amino-5-phenyl-4-pyrazole-carboxamide (202.22),1-methyl-2-benzyl-malonamate (207.33),4-amino-2,3,5,6-tetrafluorobenzamide (208.11), 2,3-napthlenedicarboxylicacid (212.22). The compounds are placed in DMSO at the concentrationdescribed above. One μl of the material is then mixed withalpha-cyano-4-hydroxy cinnamic acid matrix (after a 1:10,000 dilution)and deposited on to a solid stainless steel support.

The material is then desorbed by a laser using the Protein TOF MassSpectrometer (Bruker, Manning Park, Mass.) and the resulting ions aremeasured in both the linear and reflectron modes of operation. Thefollowing m/z values are observed (FIG. 11):

    ______________________________________                                        121.1→                                                                          benzamide (121.14)                                                     122.1→ nicotinamide (122.13)                                           123.1→ pyrazinamide (123.12)                                           124.1                                                                         125.2                                                                         127.3→ 3-amino-4-pyrazolecarboxylic acid (127.10)                      127.2→ 2-thiophenecarboxamide (127.17)                                 135.1→ 4-aminobenzamide (135.15)                                       135.1→ tolumide (135.17)                                               136.2→ 6-methylnicotinamide (136.15)                                   137.1→ 3-aminonicotinamide (137.14)                                    138,2→ nicotinamide N-oxide (138.12)                                   138.2→ 3-hydropicolinamide (138.13)                                    139.2→ 4-fluorobenzamide (139.13)                                      140.2                                                                         147.3→ cinnamamide (147.18)                                            148.2                                                                         149.2 4-methoxybenzamide (151.17)                                             152.2 2,6-difluorbenzamide (157.12)                                           158.3 4-amino-5-imidazole-carboxyamide (162.58)                               163.3                                                                         165.2→ 3,4-pyridine-dicarboxyamide (165.16)                            165.2→ 4-ethoxybenzamide (165.19)                                      166.2→ 2,3-pyrazinedicarboxamide (166.14)                              166.2→ 2-nitrobenzamide (166.14)                                        3-fluoro-4-methoxybenzoic acid (170.4)                                       171.1                                                                         172.2                                                                         173.4 indole-3-acetamide (174.2)                                              178.3                                                                         179.3→ 5-acetylsalicylamide (179.18)                                   181.2→ 3,5-dimethoxybenzamide (181.19)                                 182.2→ 1-naphthaleneacetamide (185.23)                                 186.2 8-chloro-3,5-diamino-2-pyrazinecarboxyamide (187.59)                    188.2                                                                         189.2→ 4-trifluoromethyl-benzamide (189.00)                            190.2                                                                         191.2                                                                         192.3 5-amino-5-phenyl-4-pyrazole-carboxamide (202.22)                        203.2                                                                         203.4 1-methyl-2-benzyl-malonamate (207.33)                                    4-amino-2,3,5,6-tetrafluorobenzamide (208.11)                                212.2→ 2,3-napthlenedicarboxylic acid (212.22).                        219.3                                                                         221.2                                                                         228.2                                                                         234.2                                                                         237.4                                                                         241.4                                                                       ______________________________________                                    

The data indicate that 22 of 31 compounds appeared in the spectrum withthe anticipated mass, 9 of 31 compounds appeared in the spectrum with an+H mass (1 atomic mass unit, amu) over the anticipated mass. The latterphenomenon is probably due to the protonation of an amine within thecompounds. Therefore 31 of 31 compounds are detected by MALDI MassSpectroscopy. More importantly, the example demonstrates that multipletags can be detected simultaneously by a spectroscopic method.

The alpha-cyano matrix alone (FIG. 11) gave peaks at 146.2, 164.1,172.1, 173.1, 189.1, 190.1, 191.1, 192.1, 212.1, 224.1, 228.0, 234.3.Other identified masses in the spectrum are due to contaminants in thepurchased compounds as no effort was made to further purify thecompounds.

Example 14 Assay of Gene Expression Using Multiple Probes

Sodium borate buffers (SBB) were freshly prepared from boric acid andsodium hydroxide. APB buffer is 0.18 M NaCl, 0.05 M Tris pH 7.6, 5 mMEDTA, and 0.5% Tween 20R. TMNZ buffer is 0.05 M Tris pH 9.5, 1 mM MgCl2,0.5 mM ZnCl2. FW (filter wash) is 0.09 M NaCl, 50 mM Tris pH 7.6, 25 mMEDTA. SDS/FW is FW with 0.1% sodium dodecyl sulfate (SDS). Lysis andhybridization solution is 3 M guanidinium thiocyante, 2%N-lauroylsarcosine (sarcosyl), 50 mM Tris pH 7.6 and 25 mM EDTA. CAPbuffer is 0.1 M sodium citrate and 0.2 M sodium phosphate, pH 6.5. HRP(horseradish peroxidase) substrate solution is 0.1 M sodium citrate pH6.5, 0.2 M sodium phosphate, 2.87 mM 4-methoxy-1-naphthol, 0.093 mM3-methyl-2-benzothiazolinone hydrazone hydrochloride and 4 mM hydrogenperoxide. AP (alkaline phosphatase) substrate solution is 1 mM5-bromo-4-chlorindoyl-3-phosphate, 1 mM nitroblue tetrazolium, and 0.01%Tween 20 in TMNZ. The fluorescent substrate for alkaline phosphatase is0.5 mM 4-methyl-umbelliferone phosphate, 0.05 M Tris pH 9.5, 1 mM MgCl2,0.5 mM ZnCl2. Poly(ethyleneimine) was purchased from Polysciences(Warrington, Pa.). Burnished or unpolished nylon beads were purchasedfrom The Hoover Group (Sault St. Marie, Mich.). Triethyloxoniumtetrafluoroborate, succinic anhydride and 1-methyl-2-pyrrolidinone werepurchased from Aldrich Chemical (Milwaukee, Wis.). Tween 20R andNHS-LC-Biotin were purchased from Pierce (Rockford, Ill.). Guanidinethiocyanate (GuSCN) was purchased from Kodak (Rochester, N.Y.). Cyanuricchloride was from Aldrich Chemical Co. (Milwaukee, Wis.) and wasrecrystallized from toluene.

A. ODN Synthesis

ODNs complementary (5'-CCTTAGGACAGTCTTCTTCACGC) to conserved orhypervariable regions of the 16S ribosomal RNA (rRNA) of Porphyromonasgingivalis (Pg), were synthesized on either an ABI 380B or a MilliGen7500 automated DNA synthesizer using the standardcyanoethyl-N,N-diisopropylamino-phosphoramidite (CED-phosphoramidite)chemistry. Amine tails were incorporated onto the 5'-end using thecommercially availableN-monomethoxytritylaminoihex-6-yloxy-CED-phosphoramidite. ODNs with5'-monomethoxytritryl groups were chromatographed by HPLC using aHamilton PRP-1 (7.0×305 mm) reversed-phase column employing a gradientof 5% to 45% CH3CN in 0.1 M Et3NH+OAc-, pH 7.5, over 20 min. Afterdetritylation with 80% acetic acid, the ODN s were precipitated byaddition of 3 M sodium acetate and 1-butanol. Analytical checks for thequality of the ODNs were done by ion-exchange HPLC using a Toso-HaasDEAE-NPR column and by denaturing polyacrylamide gel electrophoresis(PAGE).

B. Preparation of the Polymer-coated Nylon Bead

Unpolished nylon beads (25,000, 3/32 inch diameter) in anhydrous1-methyl-2-pyrrolidinone (1800 mL) were stirred for 5 min. at ambienttemperature. Triethyloxonium tetrafluoroborate (200 mL, 1 M indichloromethane) was added and then stirred for 30 min. at ambienttemperature. The liquid was decanted and the beads were washed quicklywith 1-methyl-2-pyrrolidinone (4×500 mL). The beads were then stirredfor 12-24 hr a 3% (w/v) solution (1 L) of 70,000 MW poly(ethyleneimine)in 1-methyl-2-pyrrolidinone (prepared from a 30% aqueous solution ofpoly(ethyleneimine)). After decanting the poly(ethyleneimine solutionthe beads were washed with 1-methyl-2-pyrrolidinone (2×1 L), SDS/FW (2×1L), H₂ O (10×2 L), and finally with 95% ethanol (1×500 mL). The beadswere dried under high vacuum for 4 to 5 h. The amine content of thebeads was determined by reaction with picrylsulfonic acid.

C. Preparation of5'-[6-(4.6-Dichloro-1,3,5-triazin-2-ylamino)-hexyl]-ODNs

To a solution of 5'-aminohexyl ODN (1 mL, 10 mg/mL) in freshly prepared0.1 M SBB (pH 8.3, 3.2 mL) and H2O (1.8 mL) was added an acetonitrilesolution of recrystallized cyanuric chloride (1 mL, 50 mg/mL). Thesolution was mixed for 30-120 minutes at ambient temperature. Theunreacted cyanuric chloride was removed by ultrafiltration through a3000 MW cutoff membrane (Amicon, Beverly, Mass.) using freshly prepared0.1 M SBB n(pH 9.3, 4×10 mL) as the wash solution. After the final washthe volume was reduced to 1 mL. The5'-[6-(4,6-dichloro-1,3,5-triazin-2-ylamino)hexyl]-ODNs are stable for 1week at 4° C. in 0.1 M SBB (pH 8.3) with no detectable decomposition.

D. Attachment of ODNs to Nylon Beads

PEI-coated nylon beads (500 beads), described above, were placed in anequal volume of freshly prepared 0.1 M SBB (pH 9.3) and vigorouslyagitated for 30 min. to rehydrate the beads. The borate solution wasdecanted and the beads were washed once with 0.1 MSBB (pH 8.3) thenvocered with an equal volume of fresh 0.1 M SBB. The borate solution ofthe 5'-[6-(4-6-dichloro-1,3,5-triazin-2-ylamino)hexyl]-ODN (1 mL, 500mg/mL) was then added to the beads. The mixture was vigorously agitatedat ambient temperature for 60 min. The solution was decanted and thebeads were then washed with 0.1 M SBB (pH 8.3, 2×500 mL). The beads weretreated in three times the volume of the beads with succinic anhydride(10 mg/mL) in 9:1 1-methyl-2-pyrrolidinone: 1.0 M SBB (pH 8.3). Thereaction mixture was stirred for 1 h at ambient temperature. The beadswere then washed with 1-methyl-2-pyrrolidinone (3×250 mL), dH2O (2×1 L),SDS/FW (5×250 mL), and then with dH2O (4×1 L). The beads were stored in25 mM EDTA.

E. Design and Labeling the Probes

In this part of the example 5 probes are designed that will permit thedifferential mRNA expression in stimulated versus unstimulated Jurkathuman T-cell lymphoma (JRT 3.5).

100 μg of each of the 5'-terminal amine-linked oligonucleotidesdescribed above are reacted with an excess recrystallized cyanuricchloride in 10% n-methyl-pyrrolidone alkaline (pH 8.3 to 8.5 preferably)buffer at 19° C. to 25° C. for 30 to 120 minutes. The final reactionconditions consist of 0.15 M sodium borate at pH 8.3, 2 mg/mlrecrystallized cyanuric chloride and 500 ug/ml respectiveoligonucleotide. The unreacted cyanuric chloride is removed by sizeexclusion chromatography on a G-50 Sephadex column. The activatedpurified oligonucleotide is then reacted with a 100-molar excess ofcystamine in 0.15 M sodium borate at pH 8.3 for 1 hour at roomtemperature. The unreacted cystamine is removed by size exclusionchromatography on a G-50 Sephadex column. The derived ODNs are thenreacted with amine-reactive fluorochromes. The derived ODN preparationis divided into 3 portions and each portion is reacted with either (a)20-fold molar excess of Texas Red sulfonyl chloride (Molecular Probes,Eugene, Oreg.), with (b) 20-fold molar excess of Lissamine sulfonylchloride (Molecular Probes, Eugene, Oreg.), (c) 20-fold molar excess offluorescein isothiocyanate. The final reaction conditions consist of0.15 M sodium borate at pH 8.3 for 1 hour at room temperature. Theunreacted fluorochromes are removed by size exclusion chromatography ona G-50 Sephadex column. IL-2, IFN-g, GM-CSF, were labelled with TexasRed. c-fos IL-4 and PKC-g were labelled with lissamine and CTLA4/CD28and GMP kinase were labelled with fluroescein. The IL-2, c-fos and CTLA4probes were pooled. The IFN-g, IL-4 and GMP kinase probes were pooledand GM-CSF and PKC-g probes were pooled.

F. Solid Support cDNA Synthesis for Gene Expression Assay

    Oligo DMO 596                                                                         5'- ACTACTGATCAGGCGCGCCTTTTTTTTTTTTTTTTTTTT -3'                          -      spacer   Asc I    (poly dT)20                                   

G. Stimulation and RNA Prep

Jurkat line JRT 3.5 is stimulated for 6 hours at a cell density of1×10e6 cells/ml in serum-free RPMI medium (Life Technologies.Gaithersburg, Md.) in the presence of 10 ng/ml phorbol-12-myristate-13acetate (Calbiochem, San Diego, Calif.) and 100 ng/ml ionomycin

(Calbiochem). Cells are pelleted, washed in 1×PBS (Life Technologies),re-pelleted and lysed in 0.5 ml, per 10⁻⁶ cells, buffer containing 4Mguanidine isothiocyanate/1% N-lauryl sarcosine/25 mM sodium citrate pH7.1 (Fisher Scientific. Pittsburg, Pa.). One-tenth volume 2M sodiumacetate (Fisher Scientific) pH 4.2 is added followed by one volume ofwater saturated phenol (Amresco. Solon, Ohio). After mixing, one-fourthvolume chloroform:isoamyl alcohol, (29:1), (Fisher Scientific) is addedand the solution is mixed vigorously, then incubated on ice for 10minutes. The lysate is then spun, the aqueous phase removed andextracted with an equal volume of chloroform:isoamyl alcohol. Theaqueous phase is then pooled and the RNA precipitated with 2 volumes ofEtOH (Quantum Chemical Corp., Tuscola, Ill.). After centrifugation, theEtOH is decanted and the RNA is air-dried briefly, then resuspended inRNase-free water to a concentration of between 1 and 5 mg/ml.

H. Capture and First Strand Synthesis

One nylon bead bearing the covalently linked oligonucleotide,5'-ACTACTGATCAGGCGCGCCTTTTTT

TTTTTTTTTTTTTT-3' (GenSet, La Jolla, Calif.), is added to, 10, ug totalcellular RNA, diluted in enough RNase-free water to cover the bead, in asterile 1.5 ml microfuge tube (Fisher Scientific). The RNA and bead areincubated at 65C for 5 minutes. An equal volume of 2× mRNA hybridizationbuffer consisting of 50, mM Tris pH 7.5 , 1M NaCl (Fisher Scientific)and 20, ug/ml acetylated-BSA (New England Biolabs, Beverly, Mass.) isadded to each tube and the tubes rocked gently for 2 hours at roomtemperature. The supernatant is removed and the bead is then washedthree times in 1× mRNA hybridization buffer. After the final wash iscomplete, a reverse transcription mix consisting of 1× MMLV-reversetranscriptase buffer, 1, mM dNTP mix, 2, mM DTT (Life Technologies), 20units Rnasin (Promega. Madison, Wis.)and 10, ug/ml acetylated-BS (NewEngland Biolabs) is added to each tube followed by addition of 600 unitsMMLV-reverse transcriptase(Life Technologies). This reaction is rockedgently at 42° C. for 2 hours. 1 unit RNase H (Boehringer-Mannheim.Indianapolis, Ind.) is then added and the reaction allowed to continuefor another 0.5 hour. The supernatant is again removed and each bead iswashed three times in 10 mM Tris pH 8.0, 1 mM EDTA pH 8(FisherScientific). Remaining RNA template is removed by boiling the beads inTE with 0.01% SDS (Fisher Scientific).

The nylon solid support was then hybridized with 100 nanograms per ml ofthe following tagged oligonucleotide probes(5'-GAACTCAAACCTCTGGAGGAAGTG-3', IL-2,5'-CAGTGCAGAGGCTCGCGAGCTATA-3',IFN-gamma 5'-CTTGACCATGATGGCCAGCCACTA-3',GM-CSF 5'-CATTCCCACGGTCACTGCCATCTC-3', c-fos5'-GCGACTGTGCTCCGGCAGTTCTAC-3', IL-4 5'-GTGGTTCATCGACGATGCCACGAA-3',PKC-gamma 5'-GAGCTCATGTACCCACCTCCGTAC-3', CTLA4/CD285'-ATCTTCGTGCAGCCGCCCTCACTG-3', GMP kinase)

(All oligos are for the human homologs except for GMP kinase which wasbased on the bovine sequence). Hybridization was in 3 m GuSCN for 8hours at 37 C. The reaction mixture was gently mixed during thehybridization to promote diffusion of the probe to the solid support.After the 8 hour incubation period, the solid support was washed twicewith 3 M GuSCN, 5 times with 0.1× SSC and then placed in 0.01 Mdithiothreitol to cleave the fluorochrome from the oligonucleotide,. Themixture is incubated for 15 minutes at room temperature. Fluorescence ismeasured in a black microtiter plate (Dynatek Laboratories, Chantilly,Va.). The plates are then read directly using a Fluoroskan IIfluorometer (Flow Laboratories, McLean, Va.) using an excitationwavelength of 495 nm and monitoring emission at 520 nm for fluorescein,using an excitation wavelength of 591 nm and monitoring emission at 612nm for Texas Red, and using an excitation wavelength of 570 nm andmonitoring emission at 590 nm for lissamine. The results from theprobing are as follows:

    ______________________________________                                                 Unstimulated   Stimulated                                            ______________________________________                                        IL-2       1.2 rfu          230 rfu                                             IFN 0.8 rfu  120 rfu                                                          GM-CSF 21 rfu  38 rfu                                                         c-fos 16 rfu  76 rfu                                                          IL-4 33 rfu  12 rfu                                                           PKC 10 rfu 130 rfu                                                            CTLA-4 ND ND                                                                  GMP kinase 450 rfu  420 rfu                                                 ______________________________________                                    

Example 15 Detection of a Single Base-Pair Mismatch on a Solid Phase

This example describes the detection of a single-base pair mismatch inan immobilized probe using complementary fluorescently labeledoligonucleotides. The set of probe oligonucleotides consists of oneprobe which forms perfect base-pairing and one oligonucleotide whichcontains the mismatch when hybridized. The two oligonucleotides arelabeled with different fluorochromes, and after hybridization is allowedto occur at the T_(m) of the mismatch, the ratio of hybridizedfluorochromes is determined.

A "target" oligonucleotide (DMO501: 5'-TTGATTCCCAATTATGCGAAGGAG-3') wasimmobilized on a set of solid supports. ODN-beads (3/32nd inch diameter)were prepared as previously described (Van Ness et al., Nucl. Acids Res.19:3345, 1991). The ODN-beads contained 0.01 to 1.2 mg/bead ofcovalently immobilized ODN. DMO578 is the complement to DMO501 (perfectcomplement). DMO1969 is the complement to DMO501 with a G→T change atposition 11. DMO1971 is the complement to DMO501 with a A→T change atposition 12. Each probe oligonucleotide was labeled with either BIODIPY,TAMRA or Texas Red. Hybridization reactions were assembled in 3 M GuSCN,0.01 M Tris pH 7.6, 5 mM EDTA at 50 ng/ml respective probe. Equal molarratios of each probe type were used in each hybridization in thepresence of 3 solid supports per tube. Hybridizations are performed at42° C. for 30 minutes with constant agitation. The beads were washedtwice with 3 M GuSCN at 42° C. and then with SDS/FW 5 times.

To denature the probe oligonucleotide, the solid supports are placed in200 μl TE (TE is 0.01 M Tris, pH 7.0, 5 mM EDTA). The mixture isincubated for 10 minutes at 100° C. Fluorescence is measured in a blackmicrotiter plate. The solution is removed from the incubation tubes (200microliters) and placed in a black microtiter plate (DynatekLaboratories, Chantilly, Va.). The plates are then read directly using aFluoroskan II fluorometer (Flow Laboratories, McLean, Va.) using anexcitation wavelength of 495 nm and monitoring emission at 520 nm forfluorescein, using an excitation wavelength of 591 nm and monitoringemission at 612 nm for Texas Red, and using an excitation wavelength of570 nm and monitoring emission at 590 nm for lissamine or TAMRA.

The results are as follows:

                  TABLE 10                                                        ______________________________________                                                  Fluorochrome ratio in                                                                        Fluorochrome ratio after                               Probe Mix hybridization mix denaturing                                      ______________________________________                                        578TR/578BD                                                                             1.9/1           1.9/1                                                 578TR/1969BD 2.0/1   25/1                                                     578TR/1971TA 0.025/1  0.58/1                                                  578BD/1971TA 0.014/1  0.48/1                                                ______________________________________                                    

The results indicate that there is no effect of the fluorochrome on thehybridization as indicated in line 1 that Texas Red (TR) 578oligonucleotide and 578-BD (BIODIPY) competed evenly for hybridizationto the immobilized target since the ratio of labels did not change afterhybridization. There is an average of a 20-fold enrichment of perfectlybased probes over the mismatched probes in GuSCN allowing certaindetection of base-pair mismatches.

Example 16

In this Example (16), all reactions were conducted in foil-coveredflasks. The sequence of reactions A→F described in this Example isillustrated in FIGS. 15A and 15B. Compound numbers as set forth in thisExample refer to the compounds of the same number in FIGS. 15A and 15B.

A. To a solution of ANP linker (compound 1, 11.2 mmol) anddiisopropylethylamine (22.4 mmol) in CHCl₃ (60 ml) was added allylbromide (22.4 mmol). The reaction mixture was refluxed for 3 hours,stirred at room temperature for 18 hours, diluted with CHCl₃ (200 ml),and washed with 1.0 M HCl (2×150 ml) and H₂ O (2×150 ml). The organicextracts were dried (MgSO₄) and the solvent evaporated to give compound2 as a yellow solid.

To a mixture of compound 2 in CH₂ Cl₂ (70 ml), tris (2-aminoethyl) amine(50 ml) was added and the reaction mixture stirred at room temperaturefor 18 hours. The reaction was diluted with CH₂ Cl₂ (150 ml) and washedwith pH 6.0 phosphate buffer (2×150 ml). The organic extracts were dried(MgSO₄) and the solvent evaporated. The residue was subjected to columnchromatography (hexane/EtOAc) to give 1.63 g (58%) of compound 3: ¹ HNMR (DMSO-d₆): δ7.85 (dd, 2H), 7.70 (t, 1H), 7.43 (t, 1H), 5.85 (m, 1H),5.20 (q, 2H), 4.58 (q, 1H), 4.50 (d, 2H), 2.70 (m, 2H), 2.20 (br s, 2H).

B. To a solution of Boc-5-aminopentanoic acid (1.09 mmol) and NMM (3.27mmol) in dry DMF (6 ml), was added HATU (1.14 mmol) and the reactionmixture stirred at room temperature for 0.5 hours. A solution ofcompound 3 (1.20 mmol) in dry DMF (1 ml) was added and the reactionmixture stirred at room temperature for 18 hours. The reaction wasdiluted with EtOAc (50 ml) and washed with 1.0 M HCl (2×50 ml) and D.I.H₂ O (2×50 ml). The organic extracts were dried (MgSO₄) and evaporatedto dryness. The residue was subjected to column chromatography to give420 mg (91%) of compound 4: ¹ H NMR (DMSO-d₆): δ8.65 (d, 1H), 7.88 (d,1H), 7.65 (m, 2H), 7.48 (t, 1H), 6.73 (br s, 1H), 5.85 (m, 1H), 5.55 (m,1H), 5.23 (q, 2H), 4.55 (d, 2H), 2.80 (m, 2H), 2.05 (t, 2H), 1.33 (s,9H), 1.20-1.30 (m, 4H).

C. A solution of compound 4 (0.9 mmol) in HCl•1,4-dioxane (20 mmol) wasstirred at room temperature for 2 hours. The reaction mixture wasconcentrated, dissolved in MeOH and toluene, and concentrated again (5×5ml) to give 398 mg (quantitative) of the compound 5: ¹ H NMR (DMSO-d₆):δ8.75 (d, 1H), 7.88 (d, 1H), 7.65 (m, 2H),7.51 (t, 1H), 7.22 (m,2H),5.85 (m, 1H), 5.57 (m, 1H), 5.23 (q, 2H), 4.55 (d, 2H), 2.80 (m,2H), 2.71 (m, 2H), 2.07 (s, 2H), 1.40-1.48 (br s, 4 H).

D. To a solution of compound 21 (0.48 mmol, prepared according toExample 18) and NMM (1.44 mmol) in dry DMF (3 ml), was added HATU (0.50mmol) and the reaction mixture stirred at room temperature for 0.5hours. A solution of compound 5 (0.51 mmol) in dry DMF (3 ml) was addedand the reaction stirred at room temperature for 18 hours. The reactionmixture was diluted with EtOAc (75 ml) and washed with 5% Na₂ CO₃ (3×50ml). The organic extracts were dried (MgSO₄) and the solvent evaporatedto give 281 mg (78%) of compound 6: ¹ H NMR (DMSO-d₆): δ8.65 (d, 1H),8.17 (d, 1H), 7.82-7.95 (m, 4H), 7.68 (m, 3H), 7.50 (t, 1H), 6.92 (d,1H), 5.85 (m, 1H), 5.57 (m, 1H), 5.20 (q, 2H), 4.55 (d, 2H), 4.30 (q,1H), 4.05 (q, 2H), 2.95 (m, 4H), 2.80 (m, 2H), 2.72 (m, 2H), 2.05 (s,3H), 2.01 (t, 2H), 1.58-1.77 (m, 3H), 1.50 (m, 4H), 1.30 (q, 3H),1.17-1.40 (m, 9H).

E. To a mixture of compound 6 (0.36 mmol) in THF (4 ml), was added 1 MNaOH (1 mmol) and the reaction stirred at room temperature for 2 hours.The reaction mixture was acidified to pH 7.0 with 1.0 M HCl (1 ml) andthe solvent evaporated to give compound 7 (quantitative): ¹ H NMR(DMSO-d₆): δ8.65 (d, 1H), 8.17 (d, 1H), 7.82-7.95 (m, 4H), 7.68 (m, 3H),7.50 (t, 1H), 6.92 (d, 1H), 5.52 (m, 1H), 4.30 (q, 1H), 4.05 (q, 2H),2.95 (m, 4H), 2.80 (m, 2H), 2.72 (m, 2H), 2.05 (s, 3H), 2.01 (t, 2H),1.58-1.77 (m, 3H), 1.50 (m, 4H), 1.30 (q, 3H), 1.17-1.40 (m, 9H).

F. To a solution of compound 7 (0.04 mmol) and NMM (0.12 mmol) in dryDMF (0.4 ml), was added HATU (0.044 mmol) and the reaction stirred atroom temperature for 0.5 hours. Allylamine (0.12 mmol) was added and thereaction mixture stirred at room temperature for 5 hours. The reactionmixture was diluted with EtOAc (15 ml) and washed with 5% Na₂ CO₃ (3×10ml). The organic extracts were dried (MgSO₄) and the solvent evaporatedto yield 15 mg (49%) of compound 8: ¹ H NMR (DMSO-d₆): δ8.49 (d, 1H),8.17 (d, 1H), 7.82-7.95 (m, 4H), 7.68 (m, 3H), 7.50 (t, 1H), 6.92 (d,1H), 5.72 (m, 1H), 5.50 (m, 1H), 5.03 (q, 2H), 4.37 (d, 2H), 4.30 (q,1H), 4.05 (q, 2H), 2.95 (m, 4H), 2.80 (m, 2H), 2.72 (m, 2H), 2.05 (s,3H), 2.01 (t, 2H), 1.58-1.77 (m, 311), 1.50 (m, 4H), 1.30 (q, 311),1.17-1.40 (m, 9H).

Example 17

The sequence of reactions A→G as described in this Example 17 isillustrated in FIGS. 16A and 16B. Compound numbers as set forth in thisExample refer to the compounds of the same number in FIGS. 16A and 16B.

A. To a solution of Fmoc-Lys(Boc)-OH (compound 9, 33.8 mmol) in CHCl₃(200 ml), was added diisopropylethylamine (67.5 mmol) and allyl bromide(67.5 mmol). The reaction mixture was refluxed for 6 hours, stirred atroom temperature for 16 hours, diluted with CHCl₃, washed with 1.0 M HCl(2×150 ml), saturated NaHCO₃ (1×150 ml) and D.I. H₂ O (2×150 ml). Theorganic extracts were dried (MgSO₄) and the solvent evaporated to yieldcompound 10.

To a solution of compound 10 in CHCl₃ (90 ml), was added pyrrolidine (10eq.) and the reaction was stirred at room temperature for 2.5 hours. Thereaction mixture was diluted with CHCl₃ (150 ml) and washed withsaturated NaHCO₃ (3×250 ml). The organic extracts were dried (MgSO₄) andthe solvent evaporated. The residue was subjected to columnchromatography (EtOAc/MeOH) to give 6.52 g (67%) of compound 11: ¹ H NMR(CDCl₃): δ5.90 (m, 1H), 5.27 (m, 2H), 4.60 (d, 2H), 3.48 (t, 1H), 3.10(d, 2H), 1.40-1.78 (m, 9H),1.40 (s, 9H).

B. To a solution of N-methylisonipecotic acid (1.60 mmol) and N-methylmorpholine (4.80 mmol) in dry DMF (5 ml), was added HATU (1.67 mmol).After 0.5 hours, a solution of compound 11 (1.75 mmol) in dry DMF (2 ml)was added and the reaction mixture stirred at room temperature for 18hours. The reaction mixture was diluted with CH₂ CL₂ (60 ml) and washedwith saturated Na₂ CO₃ (3×40 ml). The organic extracts were dried(MgSO₄) and the solvent evaporated. The residue was subjected to columnchromatography (CH₂ Cl₂ /MeOH/triethylamine) to give 580 mg (88%) ofcompound 12: ¹ H NMR (DMSO): δ8.12 (d, 1H), 6.77 (t, 1H), 5.90 (m, 1H),5.27 (m, 2H), 4.53 (d, 2H), 4.18 (m, 1H),2.62-2.90 (m, 5H), 2.13 (s,3H),1.85 (m, 2H), 1.57 (m, 5H),1.35 (s, 9H), 1.00 (t, 2H).

C. A mixture of compound 12 (1.39 mmol) in HCl•1,4-dioxane (20 mmol) wasstirred at room temperature for 4 hours. The reaction mixture wasconcentrated, dissolved in MeOH, coevaporated with toluene (5×5 ml) togive 527 mg (quantitative) of compound 13: ¹ H NMR (DMSO-d₆): δ8.12 (d,1H), 6.77 (t, 1H), 5.90 (m, 1H), 5.27 (m, 2H), 4.53 (d, 2H), 4.18 (m,1H), 2.65-3.00 (m, 8H), 2.23 (s, 3H),1.85 (m, 2H), 1.57 (m, 5H), 1.00(t, 2H).

D. To a solution of 4-ethoxybenzoic acid (1 eq.) in dry DMF, is addedNMM (3 eq.) and HATU (1.05 eq.). After 0.5 hours, a solution of compound13 in dry DMF is added. After the completion of the reaction and basicworkup, the compound 14 is isolated and purified.

E. To a solution of compound 14 in THF, is added 1N NaOH and thereaction mixture stirred at room temperature. After the completion ofthe reaction and acidification, the compound 15 is isolated.

F. To a solution of compound 15 (1 eq.) in dry DMF, is added NMM (3 eq.)and HATU (1.05 eq.). After 0.5 hours, a solution of compound 21(ANP--allyl ester, prepared according to Example 18) in dry DMF isadded. After the completion of the reaction and basic workup, the titlecompound 16 is isolated and purified.

G. To a solution of compound 16 in THF, is added 1N NaOH and thereaction mixture stirred at room temperature. After the completion ofthe reaction and acidification, the compound 17 is isolated.

Example 18

The sequence of reaction A through D as described in this Example 18 isillustrated in FIG. 16. Compound numbers as set forth in this Example,as well as Examples 16 and 17, refer to the compounds of the same numberin FIG. 17.

A. To a solution of 4-ethoxybenzoic acid (7.82 mmol) and N-methylmorpholine (20.4 mmol) in CH₂ Cl₂ (10 ml), was added HATU (7.14 mmol).After 0.25 hours, a solution of compound 11 (6.8 mmol) in CH₂ Cl₂ (6 ml)was added and the reaction mixture stirred at room temperature for 18hours. The reaction was diluted with CH₂ Cl₂ (150 ml) and washed with1.0 M HCl (3×50 ml) and saturated NaHCO₃ (3×50 ml). The organic extractswere dried (MgSO₄) and the solvent evaporated. The residue was subjectedto column chromatography (CH₂ Cl₂ /MeOH) to give 2.42 g (82%) ofcompound 18: ¹ H NMR (CDCl₃): δ7.78 (d, 2H), 6.91 (d, 2H), 6.88 (d, 1H),5.83-5.98 (m, 1H), 5.21-5.38 (m, 2H), 4.80 (q, 1H), 4.66 (d, 2H), 4.06(q, 2H), 3.11 (q, 2H), 1.90-2.04 (m, 1H), 1.68-1.87 (m, 1H), 1.39 (t,3H), 1.34 (s, 9H), 1.32-1.58 (m, 4H).

B. A mixture of compound 18 (5.5 mmol) in HCl•1,4-dioxane (14.3 mmol)was stirred at room temperature for 1 hour. The reaction mixture wasconcentrated, dissolved in MeOH, azeotroped with toluene, andconcentrated again (5× ml) to give a quantitative yield of compound 19.

C. To a solution of N-methylisonipecotic acid (6.21 mmol) in dry DMF (15mL), was added NMM (21.6 mmol) and HATU (5.67 mmol). After 0.5 hours, asolution of compound 19 (5.4 mmol) in dry DMF (10 ml) was added and thereaction stirred at room temperature for 18 hours. The reaction mixturewas brought to pH 12 with 1N NaOH (20 ml) and extracted with CHCl₃(2×200 ml). The organic extracts were dried (MgSO₄) and the solventevaporated to give 2.2 g (89%) of compound 20: ¹ H NMR (DMSO-d₆): δ8.52(d 1H), 7.84 (d, 2H), 7.72 (t, 1H), 6.95 (d, 2H), 5.80-5.95 (m, 1H),5.18-5.31 (dd, 2H), 4.58 (d, 2H), 4.37 (q, 1H), 4.08 (q, 2H), 3.01 (d,2H), 2.08 (s, 3H), 1.95 (m, 1H), 1.63-1.82 (m, 4H), 1.51 (m, 4H), 1.32(t, 3H), 1.22-1.41 (m, 6H).

D. To a solution of compound 20 (4.4 mmol) in THF (10 ml), is added 1NNaOH (4.4 mmol) and the reaction mixture stirred at room temperature for1 hour. The reaction was concentrated, dissolved in THF/toluene (2×5ml), concentrated, dissolved in CH₂ Cl₂ /toluene (1×5 ml) andconcentrated again to give a quantitative yield of compound 21: ¹ H NMR(DMSO-d₆): δ7.76 (d, 2H), 6.96 (d, 2H), 4.04 (q, 2H), 3.97 (d, 1H), 2.97(d, 2H), 2.64 (d, 2H), 2.08 (s, 3H), 1.95 (m, 1H), 1.58-1.79 (m, 4H),1.44 (m, 6H), 1.30 (t, 3H), 1.11-1.35 (m, 4H).

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

We claim:
 1. A method for detecting the binding of a first member to asecond member of a ligand pair, comprising:(a) combining a set of firsttagged members with a biological sample which may contain one or moresecond members, under conditions, and for a time sufficient to permitbinding of a first member to a second member, wherein said tag iscorrelative with a particular first member and detectable bynon-fluorescent spectrometry or potentiometry; (b) separating boundfirst and second members from unbound members; (c) cleaving said tagfrom said tagged first member; and (d) detecting said tag bynon-fluorescent spectrometry or potentiometry, and therefrom detectingthe binding of said first member to said second member.
 2. The methodaccording to claim 1 wherein said first members are bound to a solidsupport.
 3. The method according to claim 2, further comprising,subsequent to the step of separating bound first and second members,washing unbound members from said solid support.
 4. The method accordingto claim 1 wherein the detection of the tag is by mass spectrometry,infrared spectrometry, ultraviolet spectrometry, or, potentiostaticamperometry.
 5. The method according to claim 1 wherein greater than 4tagged first members are combined and wherein each tag is unique for aselected nucleic acid fragment.
 6. The method according to claim 1wherein said bound first and second members are separated from unboundmembers by a method selected from the group consisting of gelelectrophoresis, capillary electrophoresis, micro-channelelectrophoresis, HPLC, size exclusion chromatography and filtration. 7.The method according to claim 1 wherein said tagged first members arecleaved by a method selected from the group consisting of oxidation,reduction, acid-labile, base labile, enzymatic, electrochemical, heatand photolabile methods.
 8. The method according to claim 4 wherein saidtag is detected by time-of-flight mass spectrometry, quadrupole massspectrometry, magnetic sector mass spectrometry and electric sector massspectrometry.
 9. The method according to claim 4 wherein said tag isdetected by potentiostatic amperometry utilizing detectors selected fromthe group consisting of coulometric detectors and amperometricdetectors.
 10. The method according to claim 1 wherein steps b, c and dare performed in a continuous manner.
 11. The method according to claim1 wherein steps b, c and d are preformed in a continuous manner on asingle device.
 12. The method according to claim 11 wherein steps b, cand d are automated.
 13. The method according to claim 1 wherein saidfirst member is a nucleic acid molecule.
 14. The method according toclaim 1 wherein said second member is a nucleic acid molecule.
 15. Themethod according to claims 13 or 14 wherein said nucleic acid moleculeis generated by primer extension.
 16. The method according to claims 13or 14 wherein said nucleic acid molecule is generated from non-3'-taggedoligonucleotide primers.
 17. The method according to claims 13 or 14wherein said nucleic acid molecule is generated from taggeddideoxynucleotide terminators.
 18. The method according to claims 13 or14 wherein said first member is a protein, hormone or organic molecule.19. The method according to claim 18 wherein said protein is selectedfrom the group consisting of antibodies and receptors.
 20. A method foranalyzing the pattern of gene expression from a selected biologicalsample, comprising:(a) exposing nucleic acids from a biological sample;(b) combining said exposed nucleic acids with one or more selectedtagged nucleic acid probes, under conditions and for a time sufficientfor said probes to hybridize to said nucleic acids, wherein said tag iscorrelative with a particular nucleic acid probe and detectable bynon-fluorescent spectrometry or potentiomery; (c) separating hybridizedprobes from unhybridized probes; (d) cleaving said tag from said taggedfragment; and (e) detecting said tag by nonfluorescent spectrometry orpotentiometry, and therefrom determining the pattern of gene expressionof said biological sample.
 21. The method according to claim 20 whereinsaid biological sample is selected from the group consisting ofmammalian cells, bacteria and yeast.
 22. The method according to claim21 wherein said mammalian cells contain viruses.
 23. The methodaccording to claim 20 wherein said exposed nucleic acids is bound to asolid support.
 24. The method according to claim 23 wherein said solidsupport is a polymer.
 25. The method according to claim 23, furthercomprising, subsequent to the step of separating, washing the solidsupport.
 26. The method according to claim 20 wherein said hybridizedprobes are separated from unhybridized probes by a method selected fromthe group consisting of gel electrophoresis, capillary electrophoresis,micro-channel electrophoresis, HPLC, filtration and polyacrylamide gelelectrophoresis.
 27. The method according to claim 20 wherein saidtagged probes are cleaved by a method selected from the group consistingof oxidation, reduction, acid-labile, base labile, enzymatic,electrochemical, heat and photolabile methods.
 28. The method accordingto claim 20 wherein said tag is detected by a method selected from thegroup consisting of time-of-flight mass spectrometry, quadrupole massspectrometry, magnetic sector mass spectrometry and electric sector massspectrometry.
 29. The method according to claim 20 wherein said tag isdetected by potentiostatic amperometry utilizing detectors selected fromthe group consisting of coulometric detectors and amperometricdetectors.
 30. The method according to claim 20 wherein steps c, d and eare performed in a continuous manner.
 31. The method according to claim20 wherein steps c, d and e are performed in a continuous manner on asingle device.
 32. The method according to claim 31 wherein said deviceis automated.
 33. A compound of the formula:

    T.sup.ms -L-X

wherein T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; Lis an organic group which allows a T^(ms) -containing moiety to becleaved from the remainder of the compound, wherein the T^(ms)-containing moiety comprises a functional group which supports a singleionized charge state when the compound is subjected to mass spectrometryand is selected from tertiary amine, quaternary amine and organic acid,the compound having a mass of at least 250 daltons; and -L-X has theformula: ##STR21## wherein one or more of positions b, c, d or e issubstituted with hydrogen, alkyl, alkoxy, fluoride, chloride, hydroxyl,carboxylate or amide; R¹ is hydrogen or hydrocarbyl, and R² comprises amolecule of interest (MOI) other than a nucleic acid fragment.
 34. Acompound of the formula:

    T.sup.ms -L-X

wherein T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine, andhas the formula:

    T.sup.2 -(J-T.sup.3 -).sub.n -

wherein T² is an organic moiety formed from carbon and one or more ofhydrogen, fluoride, iodide, oxygen, nitrogen, sulfur and phosphorus,having a mass of 15 to 500 daltons; T³ is an organic moiety formed fromcarbon and one or more of hydrogen, fluoride, iodide, oxygen, nitrogen,sulfur and phosphorus, having a mass of 50 to 1000 daltons; J is adirect bond or a functional group selected from amide, ester, amine,sulfide, ether, thioester, disulfide, thioether, urea, thiourea,carbamate, thiocarbamate, Schiff base, reduced Schiff base, imine,oxime, hydrazone, phosphate, phosphonate, phosphoramide, phosphonamide,sulfonate, sulfonamide or carbon-carbon bond; and n is an integerranging from 1 to 50, and when n is greater than 1, each T³ and J isindependently selected; L is an organic group which allows a T^(ms)-containing moiety to be cleaved from the remainder of the compound,wherein the T^(ms) -containing moiety comprises a functional group whichsupports a single ionized charge state when the compound is subjected tomass spectrometry and is selected from tertiary amine, quaternary amineand organic acid; and X comprises a molecules of interest (MOI) otherthan a nucleic acid fragment, and the compound has a mass of at least250 daltons.
 35. A compound according to claim 34 wherein T² is selectedfrom hydrocarbyl, hydrocarbyl-O-hydrocarbylene,hydrocarbyl-S-hydrocarbylene, hydrocarbyl-NH-hydrocarbylene,hydrocarbyl-amide-hydrocarbylene, N-(hydrocarbyl)hydrocarbylene,N,N-di(hydrocarbyl)hydrocarbylene, hydrocarbylacyl-hydrocarbylene,heterocyclylhydrocarbyl wherein the heteroatom(s) are selected fromoxygen, nitrogen, sulfur and phosphorus, substitutedheterocyclylhydrocarbyl wherein the heteroatom(s) are selected fromoxygen, nitrogen, sulfur and phosphorus and the substituents areselected from hydrocarbyl, hydrocarbyl-O-hydrocarbylene,hydrocarbyl-NH-hydrocarbylene, hydrocarbyl-S-hydrocarbylene,N-(hydrocarbyl)hydrocarbylene, N,N-di(hydrocarbyl)hydrocarbylene andhydrocarbylacyl-hydrocarbylene, as well as derivatives of any of theforegoing wherein one or more hydrogens is replaced with an equal numberof fluorides.
 36. A compound according to claim 43 wherein T³ has theformula -G(R²)-, G is C₁₋₆ alkylene having a single R² substituent, andR² is selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl-fusedcycloalkyl, cycloalkenyl, aryl, aralkyl, aryl-substituted alkenyl oralkynyl, cycloalkyl-substituted alkyl, cycloalkenyl-substitutedcycloalkyl, biaryl, alkoxy, alkenoxy, alkynoxy, aralkoxy,aryl-substituted alkenoxy or alkynoxy, alkylamino, alkenylamino oralkynylamino, aryl-substituted alkylamino, aryl-substituted alkenylaminoor alkynylamino, aryloxy, arylamino, N-alkylurea-substituted alkyl,N-arylurea-substituted alkyl, alkylcarbonylamino-substituted alkyl,aminocarbonyl-substituted alkyl, heterocyclyl, heterocyclyl-substitutedalkyl, heterocyclyl-substituted amino, carboxyalkyl substituted aralkyl,oxocarbocyclyl-fused aryl and heterocyclylalkyl; cycloalkenyl,aryl-substituted alkyl and, aralkyl, hydroxy-substituted alkyl,alkoxy-substituted alkyl, aralkoxy-substituted alkyl, alkoxy-substitutedalkyl, aralkoxy-substituted alkyl, amino-substituted alkyl,(aryl-substituted alkyloxycarbonylamino)-substituted alkyl,thiol-substituted alkyl, alkylsulfonyl-substituted alkyl,(hydroxy-substituted alkylthio)-substituted alkyl,thioalkoxy-substituted alkyl, hydrocarbylacylamino-substituted alkyl,heterocyclylacylamino-substituted alkyl,hydrocarbyl-substituted-heterocyclylacylamino-substituted alkyl,alkylsulfonylamino-substituted alkyl, arylsulfonylamino-substitutedalkyl, morpholino-alkyl, thiomorpholino-alkyl, morpholinocarbonyl-substituted alkyl, thiomorpholinocarbonyl-substituted alkyl,[N-(alkyl, alkenyl or alkynyl)- or N,N-[dialkyl, dialkenyl, dialkynyl or(alkyl, alkenyl)-amino]carbonyl-substituted alkyl,heterocyclylaminocarbonyl, heterocylylalkyleneaminocarbonyl,heterocyclylaminocarbonyl-substituted alkyl,heterocylylalkyleneaminocarbonyl-substituted alkyl,N,N-[dialkyl]alkyleneaminocarbonyl,N,N-[dialkyl]alkyleneaminocarbonyl-substituted alkyl, alkyl-substitutedheterocyclylcarbonyl, alkyl-substituted heterocyclylcarbonyl-alkyl,carboxyl-substituted alkyl, dialkylamino-substituted acylaminoalkyl andamino acid side chains selected from arginine, asparagine, glutamine,S-methyl cysteine, methionine and corresponding sulfoxide and sulfonederivatives thereof, glycjne, leucine, isoleucine, allo-isoleucine,tert-leucine, norleucine, phenylalanine, tyrosine, tryptophan, proline,alanine, omithine, histidine, glutamine, valine, threonine, serine,aspartic acid, beta-cyanoalanine, and allothreonine; alynyl andheterocyclylcarbonyl, aminocarbonyl, amido, mono- ordialkylaminocarbonyl, mono- or diarylaminocarbonyl,alkylarylaminocarbonyl, diarylaminocarbonyl, mono- ordiacylaminocarbonyl, aromatic or aliphatic acyl, alkyl optionallysubstituted by substituents selected from amino, carboxy, hydroxy,mercapto, mono- or dialkylamino, mono- or diarylamino, alkylarylamino,diarylamino, mono- or diacylamino, alkoxy, alkenoxy, aryloxy,thioalkoxy, thioalkenoxy, thioalkynoxy, thioaryloxy and heterocyclyl.37. A compound of the formula:

    T.sup.ms -L-X

wherein T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; Lis an organic group which allows a T^(ms) -containing moiety to becleaved from the remainder of the compound, wherein the T^(ms)-containing moiety comprises a functional group which supports a singleionized charge state when the compound is subjected to mass spectrometryand is selected from tertiary amine, quaternary amine and organic acid;X comprises a molecule of interest (MOI) other than a nucleic acidfragment, the compound having a mass of at least 250 daltons; thecompound also having the formula: ##STR22## wherein G is (CH₂)₁₋₆wherein a hydrogen on one and only one of the CH, groups is replacedwith-(CH₂)_(c) -Amide-T⁴ ; T² and T⁴ are organic moieties of the formulaC₁₋₂₅ N₀₋₉ O₀₋₉ H.sub.α F.sub.β wherein the sum of α and β is sufficientto satisfy the otherwise unsatisfied valencies of the C, N, and O atoms;##STR23## R¹ is hydrogen or C₁₋₁₀ alkyl; c is an integer ranging from 0to 4; and n is an integer ranging from 1 to 50 such that when n isgreater than 1, then G, c, Amide, R¹ and T⁴ are independently selected.38. A compound of the formula:

    T.sup.ms -L-X

wherein T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; Lis an organic group which allows a T^(ms) -containing moiety to becleaved from the remainder of the compound, wherein the T^(ms)-containing moiety comprises a functional group which supports a singleionized charge state when the compound is subjected to mass spectrometryand is selected from tertiary amine, quaternary amine and organic acid;X comprises a molecule of interest (MOI) other than a nucleic acidfragment, the compound having a mass of at least 250 daltons, thecompound also having the formula: ##STR24## wherein G is (CH₂)₁₋₆wherein a hydrogen on one and only one of the CH₂ groups is replacedwith-(CH₂)_(c) -Amide-T⁴ ; T² and T⁴ are organic moieties of the formulaC¹⁻²⁵ N₀₋₉ O₀₋₉ H.sub.α F.sub.β wherein the sum of α and β is sufficientto satisfy the otherwise unsatisfied valencies of the C, N, and O atoms;##STR25## R¹ is hydrogen or C₁₋₁₀ alkyl c is an integer ranging from 0to 4; and T⁵ is an organic moiety of the formula C₁₋₂₅ N₀₋₉ O₀₋₉ H.sub.αF.sub.β wherein the sum of α and β is sufficient to satisfy theotherwise unsatisfied valencies of the C, N, and O atoms; and T⁵includes a tertiary or quaternary amine or an organic acid; and m is aninteger ranging from 0-49 where when m is greater than 1, then G, c,Amide, R¹ and T⁴ are independently selected.
 39. A compound of theformula:

    T.sup.ms -L-X

wherein T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; Lis an organic group which allows a T^(ms) -containing moiety to becleaved from the remainder of the compound, wherein the T^(ms)-containing moiety comprises a functional group which supports a singleionized charge state when the compound is subjected to mass spectrometryand is selected from tertiary amine, quaternary amine and organic acid;X comprises a molecule of interest (MOI) other than a nucleic acidfragment, the compound having a mass of at least 250 daltons; thecompound also having the formula: ##STR26## wherein G is (CH₂)₁₋₆wherein a hydrogen on one and only one of the CH₂ groups is replacedwith-(CH₂)_(c) -Amide-T⁴ ; T² and T⁴ are organic moieties of the formulaC₁₋₂₅ N₀₋₉ O₀₋₉ H.sub.α F.sub.β wherein the sum of α and β is sufficientto satisfy the otherwise unsatisfied valencies of the C, N, and O atoms;##STR27## R¹ is hydrogen or C₁₋₁₀ alkyl; c is an integer ranging from 0to 4; and T⁵ is an organic moiety of the formula C₁₋₂₅ N₀₋₉ O₀₋₉ H.sub.αF.sub.β wherein the sum of α and β is sufficient to satisfy theotherwise unsatisfied valencies of the C, N, and O atoms; and T⁵includes a tertiary or quaternary amine or an organic acid; and m is aninteger ranging from 0-49 where when m is greater than 1, then G, c,Amide, R¹ and T⁴ are independently selected.
 40. A compound according toany one of claims 38 or 39 wherein -Amide-T⁵ is selected from: ##STR28##41. A compound according to any of claims 38 or 39 wherein -Amide-T⁵ isselected from:
 42. A compound according to claim 34 wherein T² has thestructure which results when one of the following organic acids iscondensed with an amine group to form T² -C(═O)--N(R¹)--: Formic acid,Acetic acid, Propiolic acid, Propionic acid, Fluoroacetic acid,2-Butynoic acid, Cyclopropanecarboxylic acid, Butyric acid,Methoxyacetic acid, Difluoroacetic acid, 4-Pentynoic acid,Cyclobutanecarboxylic acid, 3,3-Dimethylacrylic acid, Valeric acid,N,N-Dimethylglycine, N-Formyl-Gly-OH, Ethoxyacetic acid,(Methylthio)acetic acid, Pyrrole-2-carboxylic acid, 3-Furoic acid,Isoxazole-5-carboxylic acid, trans-3-Hexenoic acid, Trifluoroaceticacid, Hexanoic acid, Ac-Gly-OH, 2-Hydroxy-2-methylbutyric acid, Benzoicacid, Nicotinic acid, 2-Pyrazinecarboxylic acid,1-Methyl-2-pyrrolecarboxylic acid, 2-Cyclopentene-1-acetic acid,Cyclopentylacetic acid, (S)-(-)-2-Pyrrolidone-5-carboxylic acid,N-Methyl-L-proline, Heptanoic acid, Ac-b-Ala-OH,2-Ethyl-2-hydroxybutyric acid, 2-(2-Methoxyethoxy)acetic acid, p-Toluicacid, 6-Methylnicotinic acid, 5-Methyl-2-pyrazinecarboxylic acid,2,5-Dimethylpyrrole-3-carboxylic acid, 4-Fluorobenzoic acid,3,5-Dimethylisoxazole-4-carboxylic acid, 3-Cyclopentylpropionic acid,Octanoic acid, N,N-Dimethylsuccinamic acid, Phenylpropiolic acid,Cinnamic acid, 4-Ethylbenzoic acid, p-Anisic acid,1,2,5-Trimethylpyrrole-3-carboxylic acid, 3-Fluoro-4-methylbenzoic acid,Ac-DL-Propargylglycine, 3-(Trifluoromethyl)butyric acid,1-Piperidinepropionic acid, N-Acetylproline, 3,5-Difluorobenzoic acid,Ac-L-Val-OH, Indole-2-carboxylic acid, 2-Benzofurancarboxylic acid,Benzotriazole-5-carboxylic acid, 4-n-Propylbenzoic acid,3-Dimethylaminobenzoic acid, 4-Ethoxybenzoic acid, 4-(Methylthio)benzoicacid, N-(2-Furoyl)glycine, 2-(Methylthio)nicotinic acid,3-Fluoro-4-methoxybenzoic acid, Tfa-Gly-OH, 2-Napthoic acid, Quinaldicacid, Ac-L-Ile-OH, 3-Methylindene-2-carboxylic acid,2-Quinoxalinecarboxylic acid, 1-Methylindole-2-carboxylic acid,2,3,6-Trifluorobenzoic acid, N-Formyl-L-Met-OH,2-[2-(2-Methoxyethoxy)ethoxy]acetic acid, 4-n-Butylbenzoic acid,N-Benzoylglycine, 5-Fluoroindole-2-carboxylic acid, 4-n-Propoxybenzoicacid, 4-Acetyl-3,5-dimethyl-2-pyrrolecarboxylic acid,3,5-Dimethoxybenzoic acid, 2,6-Dimethoxynicotinic acid,Cyclohexanepentanoic acid, 2-Naphthylacetic acid,4-(1H-Pyrrol-1-yl)benzoic acid, Indole-3-propionic acid,m-Trifluoromethylbenzoic acid, 5-Methoxyindole-2-carboxylic acid,4-Pentylbenzoic acid, Bz-b-Ala-OH, 4-Diethylaminobenzoic acid,4-n-Butoxybenzoic acid, 3-Methyl-5-CF3-isoxazole-4-carboxylic acid,(3,4-Dimethoxyphenyl)acetic acid, 4-Biphenylcarboxylic acid,Pivaloyl-Pro-OH, Octanoyl-Gly-OH, (2-Naphthoxy)acetic acid,Indole-3-butyric acid, 4-(Trifluoromethyl)phenylacetic acid,5-Methoxyindole-3-acetic acid, 4-(Trifluoromethoxy)benzoic acid,Ac-L,-Phe-OH, 4-Pentyloxybenzoic acid, Z-Gly-OH,4-Carboxy-N-(fur-2-ylmethyl)pyrrolidin-2-one, 3,4-Diethoxybenzoic acid,2,4-Dimethyl-5-CO₂ Et-pyrrole-3-carboxylic acid,N-(2-Fluorophenyl)succinamic acid, 3,4,5-Trimethoxybenzoic acid,N-Phenylanthranilic acid, 3-Phenoxybenzoic acid, Nonanoyl-Gly-OH,2-Phenoxypyridine-3-carboxylic acid,2,5-Dimethyl-1-phenylpyrrole-3-carboxylic acid,trans-4-(Trifluoromethyl)cinnamic acid,(5-Methyl-2-phenyloxazol-4-yl)acetic acid, 4-(2-Cyclohexenyloxy)benzoicacid, 5-Methoxy-2-methylindole-3-acetic acid, trans-4-Cotininecarboxylicacid, Bz-5-Aminovaleric acid, 4-Hexyloxybenzoic acid,N-(3-Methoxyphenyl)succinamic acid, Z-Sar-OH,4-(3,4-Dimethoxyphenyl)butyric acid, Ac-o-Fluoro-DL-Phe-OH,N-(4-Fluorophenyl)glutaramic acid, 4'-Ethyl-4-biphenylcarboxylic acid,1,2,3,4-Tetrahydroacridinecarboxylic acid, 3-Phenoxyphenylacetic acid,N-(2,4-Difluorophenyl)succinamic acid, N-Decanoyl-Gly-OH,(+)-6-Methoxy-a-methyl-2-naphthaleneacetic acid,3-(Trifluoromethoxy)cinnamic acid, N-Formyl-DL-Trp-OH,(R)-(+)-a-Methoxy-a-(trifluoromethyl)phenylacetic acid, Bz-DL-Leu-OH,4-(Trifluoromethoxy)phenoxyacetic acid, 4-Heptyloxybenzoic acid,2,3,4-Trimethoxycinnamic acid, 2,6-Dimethoxybenzoyl-Gly-OH,3-(3,4,5-Trimethoxyphenyl)propionic acid,2,3,4,5,6-Pentafluorophenoxyacetic acid,N-(2,4-Difluorophenyl)glutaramic acid, N-Undecanoyl-Gly-OH,2-(4-Fluorobenzoyl)benzoic acid, 5-Trifluoromethoxyindole-2-carboxylicacid, N-(2,4-Difluorophenyl)diglycolamic acid, Ac-L-Trp-OH,Tfa-L-Phenylglycine-OH, 3-Iodobenzoic acid,3-(4-n-Pentylbenzoyl)propionic acid, 2-Phenyl-4-quinolinecarboxylicacid, 4-Octyloxybenzoic acid, Bz-L-Met-OH, 3,4,5-Triethoxybenzoic acid,N-Lauroyl-Gly-OH, 3,5-Bis(trifluoromethyl)benzoic acid,Ac-5-Methyl-DL-Trp-OH, 2-Iodophenylacetic acid, 3-Iodo-4-methylbenzoicacid, 3-(4-n-Hexylbenzoyl)propionic acid, N-Hexanoyl-1-Phe-OH,4-Nonyloxybenzoic acid, 4'-(Trifluoromethyl)-2-biphenylcarboxylic acid,Bz-L-Phe-OH, N-Tridecanoyl-Gly-OH, 3,5-Bis(trifluoromethyl)phenylaceticacid, 3-(4-n-Heptylbenzoyl)propionic acid, N-Hepytanoyl-L-Phe-OH,4-Decyloxybenzoic acid, N-(α,α,α-trifluoro-m-tolyl)anthranilic acid,Niflumic acid, 4-(2-Hydroxyhexafluoroisopropyl)benzoic acid,N-Myristoyl-Gly-OH, 3-(4-n-Octylbenzoyl)propionic acid,N-Octanoyl-L-Phe-OH, 4-Undecyloxybenzoic acid,3-(3,4,5-Trimethoxyphenyl)propionyl-Gly-OH, 8-Iodonaphthoic acid,N-Pentadecanoyl-Gly-OH, 4-Dodecyloxybenzoic acid, N-Palmitoyl-Gly-OH,and N-Stearoyl-Gly-OH.
 43. A compound according to claim 33 whereinT^(ms) has a mass of from to 10,000 daltons and a molecular formula ofC₁₋₅₀₀ N₀₋₁₀₀ O₀₋₁₀₀ S₀₋₁₀ P₀₋₁₀ H.sub.α F.sub.β I.sub.δ wherein the sumof α, β and δ is sufficient to satisfy the otherwise unsatisfiedvalencies of the C, N and O atoms.
 44. A compound according to claim 33wherein T^(ms) and L are bonded together through a functional groupselected from amide, ester, ether, amine, sulfide, thioester, disulfide,thioether, urea, thiourea, carbamate, thiocarbamate, Schiff base,reduced Schiff base, imine, oxime, hydrazone, phosphate, phosphonate,phosphoramide, phosphonamide, sulfonate, sulfonamide or carbon--carbonbond.
 45. A compound according to claim 44 wherein the functional groupis selected from amide, ester, amine, urea and carbamate.
 46. A compoundaccording to claims 34, 37, 38 or 39 wherein L is slected from L^(h)υ,L^(acid), L^(base), L.sup.[O], L.sup.[R], L^(enz), L^(elc), L.sup.Δ andL^(ss), where actinic radiation, acid, base, oxidation, reduction,enzyme, electrochemical, thermal and thiol exchange, respectively, causethe T^(ms) -containing moiety to be cleaved from the remainder of themolecule.
 47. A compound according to claim 46 wherein L^(h)υ has theformula L¹ -L² -L³, wherein L² is a molecular fragment that absorbsactinic radiation to promote the cleavage of T^(ms) from X, and L¹ andL³ are independently a direct bond or an organic moiety, where L¹separates L² from T^(ms) and L³ separates L² from X, and neither L¹ norL³ undergo bond cleavage when L² absorbs the actinic radiation.
 48. Acompound according to claim 47 wherein -L² -L³ has the formula: with onecarbon atom at positions a, b, c, d or e being substituted with -L³ -Xand optionally one or more of positions b, c, d or e being substitutedwith alkyl, alkoxy, fluoride, chloride, hydroxyl, carboxylate or amide;and R¹ is hydrogen or hydrocarbyl.
 49. A compound according to claim 48wherein X comprises a molecule of interest (MOI) selected from protein,peptide, oligosaccharide, antibody, antigen, drugs and synthetic organicmolecules.
 50. A compound according to claim 47 wherein L³ is selectedfrom a direct bond, a hydrocarbylene, -O-hydrocarbylene, andhydrocarbylene-(O-hydrocarbylene)_(n) -H, and n is an integer rangingfrom 1 to
 10. 51. A compound according to claim 33 wherein X comprises amolecule of interest (MOI) selected from protein, peptide,oligosaccharide, antibody, antigen, drugs and synthetic organicmolecules.
 52. A compound according to claim 34 wherein X comprises amolecule of interest (MOI) selected from protein, peptide,oligosaccharide, antibody, antigen, drugs and synthetic organicmolecules.
 53. A compound according to claim 37 wherein X comprises amolecule of interest (MOI) selected from protein, peptide,oligosaccharide, antibody, antigen, drugs and synthetic organicmolecules.
 54. A compound according to claim 38 wherein X comprises amolecule of interest (MOI) selected from protein, peptide,oligosaccharide, antibody, antigen, drugs and synthetic organicmolecules.
 55. A compound according to claim 39 wherein X comprises amolecule of interest (MOI) selected from protein, peptide,oligosaccharide, antibody, antigen, drugs and synthetic organicmolecules.
 56. The method of claim 1 wherein the tagged first member isa compound having a mass of at least 250 daltons of the formula:

    T.sub.ms -L-X

wherein, T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; Lis an organic group which allows a T^(ms) -containing moiety to becleaved from the remainder of the compound, wherein the T^(ms)-containing moiety comprises a functional group which supports a singleionized charge state when the compound is subjected to mass spectrometryand is selected from tertiary amine, quaternary amine and organic acid;and X comprises a molecule of interest.
 57. The method of claim 1wherein the tagged first member is a compound having a mass of at least250 daltons of the formula:

    T.sup.ms -L-X

wherein T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; Lis an organic group which allows a T^(ms) -containing moiety to becleaved from the remainder of the compound, wherein the T^(ms)-containing moiety comprises a functional group which supports a singleionized charge state when the compound is subjected to mass spectrometryand is selected from tertiary amine, quaternary amine and organic acid;and -L-X has the formula: ##STR29## wherein one or more of positions b,c, d or e is substituted with hydrogen, alkyl, alkoxy, fluoride,chloride, hydroxyl, carboxylate or amide; R¹ is hydrogen or hydrocarbyl;and R² comprises a molecule of interest (MOI).
 58. The method of claim 1wherein the tagged first member is a compound having a mass of at least250 daltons of the formula:

    T.sup.ms -L-X

wherein T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine, andhas the formula:

    T.sup.2 -(J-T.sup.3 -).sub.n -

wherein T² is an organic moiety formed from carbon and one or more ofhydrogen, fluoride, iodide, oxygen, nitrogen, sulfur and phosphorus,having a mass of 15 to 500 daltons; T³ is an organic moiety formed fromcarbon and one or more of hydrogen, fluoride, iodide, oxygen, nitrogen,sulfur and phosphorus, having a mass of 50 to 1000 daltons; J is adirect bond or a functional group selected from amide, ester, amine,sulfide, ether, thioester, disulfide, thioether, urea, thiourea,carbamate, thiocarbamate, Schiff base, reduced Schiff base, imine,oxime, hydrazone, phosphate, phosphonate, phosphoramide, phosphonamide,sulfonate, sulfonamide or carbon-carbon bond; and n is an integerranging from 1 to 50, and when n is greater than 1, each T³ and J isindependently selected; and L is an organic group which allows a T^(ms)-containing moiety to be cleaved from the remainder of the compound,wherein the T^(ms) -containing moiety comprises a functional group whichsupports a single ionized charge state when the compound is subjected tomass spectrometry and is selected from tertiary amine, quaternary amineand organic acid; and X is a molecule of interest (MOI).
 59. The methodof claim 1 wherein the tagged first member is a compound having a massof at least 250 daltons of the formula:

    T.sup.ms -L-X

wherein T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; Lis an organic group which allows a T^(ms) -containing moiety to becleaved from the remainder of the compound, wherein the T^(ms)-containing moiety comprises a functional group which supports a singleionized charge state when the compound is subjected to mass spectrometryand is selected from tertiary amine, quaternary amine and organic acid;X is molecule of interest (MOI); the compound also having the formula:##STR30## wherein G is (CH₂)₁₋₆ wherein a hydrogen on one and only oneof the CH₂ groups is replaced with-(CH₂)_(c) -Amide-T⁴ ; T² and T⁴ areorganic moieties of the formula C₁₋₂₅ N₀₋₉ O₀₋₉ H.sub.α F.sub.β whereinthe sum of α and β is sufficient to satisfy the otherwise unsatisfiedvalencies of the C, N, and O atoms; ##STR31## R¹ is hydrogen or C₁₋₁₀alkyl; c is an integer ranging from 0 to 4; and n is an integer rangingfrom 1 to 50 such that when n is greater than 1, then G, c, Amide, R¹and T⁴ are independently selected.
 60. The method of claim 1 wherein thetagged first member is a compound having a mass of at least 250 daltonsof the formula:

    T.sup.ms -L-X

wherein T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; Lis an organic group which allows a T^(ms) -containing moiety to becleaved from the remainder of the compound, wherein the T^(ms)-containing moiety comprises a functional group which supports a singleionized charge state when the compound is subjected to mass spectrometryand is selected from tertiary amine, quaternary amine and organic acid;X is a molecule of interest (MOI), the compound also having the formula:##STR32## wherein G is (CH₂)₁₋₆ wherein a hydrogen on one and only oneof the CH₂ groups is replaced with-(CH₂)_(c) -Amide-T⁴ ; T² and T⁴ areorganic moieties of the formula C₁₋₂₅ N₀₋₉ O₀₋₉ H.sub.α F.sub.β whereinthe sum of (x and P is sufficient to satisfy the otherwise unsatisfiedvalencies of the C, N, and O atoms; ##STR33## R¹ is hydrogen or C₁₋₁₀alkyl; c is an integer ranging from 0 to 4; and T⁵ is an organic moietyof the formula C₁₋₂₅ N₀₋₉ O₀₋₉ H.sub.α F.sub.β wherein the sum of α andβ is sufficient to satisfy the otherwise unsatisfied valencies of the C,N, and O atoms; and T⁵ includes a tertiary or quaternary amine or anorganic acid; and m is an integer ranging from 0-49 where when m isgreater than 1, then G, c, Amide, R¹ and T⁴ are independently selected.61. The method of claim 1 wherein the tagged first member is a compoundhaving a mass of at least 250 daltons of the formula:

    T.sup.ms -L-X

wherein T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; Lis an organic group which allows a T^(ms) -containing moiety to becleaved from the remainder of the compound, wherein the T^(ms)-containing moiety comprises a functional group which supports a singleionized charge state when the compound is subjected to mass spectrometryand is selected from tertiary amine, quaternary amine and organic acid;X is a molecule of interest (MOI); the compound also having the formula:##STR34## wherein G is (CH₂)₁₋₆ wherein a hydrogen on one and only oneof the CH₂ groups is replaced with-(CH₂)_(c) -Amide-T⁴ ; T² and T⁴ areorganic moieties of the formula C₁₋₂₅ N₀₋₉ O₀₋₉ H.sub.α F.sub.β whereinthe sum of α and β is sufficient to satisfy the otherwise unsatisfiedvalencies of the C, N, and O atoms; ##STR35## R¹ is hydrogen or C₁₋₁₀alkyl; c is an integer ranging from 0 to 4; and T⁵ is an organic moietyof the formula C₁₋₂₅ N₀₋₉ O₀₋₉ H.sub.α F.sub.β wherein the sum of α andβ is sufficient to satisfy the otherwise unsatisfied valencies of the C,N, and O atoms; and T⁵ includes a tertiary or quaternary amine or anorganic acid; and m is an integer ranging from 0-49 where when m isgreater than 1, then G, c, Amide, R¹ and T⁴ are independently selected.62. The method of claim 20 wherein the tagged nucleic acid probe is acompound having a mass of at least 250 daltons of the formula:

    T.sup.ms -L-X

wherein, T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; Lis an organic group which allows a T^(ms) -containing moiety to becleaved from the remainder of the compound, wherein the T^(ms)-containing moiety comprises a functional group which supports a singleionized charge state when the compound is subjected to mass spectrometryand is selected from tertiary amine, quaternary amine and organic acid;and X comprises a nucleic acid probe.
 63. The method of claim 20 whereinthe tagged nucleic acid probe is a compound having a mass of at least250 daltons of the formula:

    T.sup.ms -L-X

wherein T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; Lis an organic group which allows a T^(ms) -containing moiety to becleaved from the remainder of the compound, wherein the T^(ms)-containing moiety comprises a functional group which supports a singleionized charge state when the compound is subjected to mass spectrometryand is selected from tertiary amine, quaternary amine and organic acid;and -L-X has the formula: ##STR36## wherein one or more of positions b,c, d or e is substituted with hydrogen, alkyl, alkoxy, fluoride,chloride, hydroxyl, carboxylate or amide; R¹ is hydrogen or hydrocarbyl;and R² comprises a nucleic acid probe.
 64. The method of claim 20wherein the tagged nucleic acid probe is a compound having a mass of atleast 250 daltons of the formula:

    T.sup.ms -L-X

wherein T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine, andhas the formula:

    T.sup.2 -(J-T.sup.3 -).sub.n -

wherein T² is an organic moiety formed from carbon and one or more ofhydrogen, fluoride, iodide, oxygen, nitrogen, sulfur and phosphorus,having a mass of 15 to 500 daltons, T³ is an organic moiety formed fromcarbon and one or more of hydrogen, fluoride, iodide, oxygen, nitrogen,sulfur and phosphorus, having a mass of 50 to 1000 daltons; J is adirect bond or a functional group selected from amide, ester, amine,sulfide, ether, thioester, disulfide, thioether, urea, thiourea,carbamate, thiocarbamate, Schiff base, reduced Schiff base, imine,oxime, hydrazone, phosphate, phosphonate, phosphoramide, phosphonamide,sulfonate, sulfonamide or carbon-carbon bond; and n is an integerranging from 1 to 50, and when n is greater than 1, each T³ and J isindependently selected; L is an organic group which allows a T^(ms)-containing moiety to be cleaved from the remainder of the compound,wherein the T^(ms) -containing moiety comprises a functional group whichsupports a single ionized charge state when the compound is subjected tomass spectrometry and is selected from tertiary amine, quaternary amineand organic acid; and X comprises a nucleic acid probe.
 65. The methodof claim 20 wherein the tagged nucleic acid probe is a compound having amass of at least 250 daltons of the formula:

    T.sup.ms -L-X

wherein T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; Lis an organic group which allows a T^(ms) -containing moiety to becleaved from the remainder of the compound, wherein the T^(ms)-containing moiety comprises a functional group which supports a singleionized charge state when the compound is subjected to mass spectrometryand is selected from tertiary amine, quaternary amine and organic acid;X comprises a nucleic acid probe; the compound also having the formula:##STR37## wherein G is (CH₂)₁₋₆ wherein a hydrogen on one and only oneof the CH₂ groups is replaced with-(CH₂)_(c) -Amide-T⁴ ; T² and T⁴ areorganic moieties of the formula C₁₋₂₅ N₀₋₉ O₀₋₉ H.sub.α F.sub.β whereinthe sum of α and β is sufficient to satisfy the otherwise unsatisfiedvalencies of the C, N, and O atoms; ##STR38## R¹ is hydrogen or C₁₋₁₀alkyl; c is an integer ranging from 0 to 4; and n is an integer rangingfrom 1 to 50 such that when n is greater than 1, then G, c, Amide, R¹and T⁴ are independently selected.
 66. The method of claim 20 whereinthe tagged nucleic acid probe is a compound having a mass of at least250 daltons of the formula:

    T.sup.ms -L-X

wherein T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; Lis an organic group which allows a T^(ms) -containing moiety to becleaved from the remainder of the compound, wherein the T^(ms)-containing moiety comprises a functional group which supports a singleionized charge state when the compound is subjected to mass spectrometryand is selected from tertiary amine, quaternary amine and organic acid;X comprises a nucleic acid probe, the compound also having the formula:##STR39## wherein G is (CH₂)₁₋₆ wherein a hydrogen on one and only oneof the CH₂ groups is replaced with-(CH₂)_(c) -Amide-T⁴ ; T² and T⁴ areorganic moieties of the formula C₁₋₂₅ N₀₋₉ O₀₋₉ H.sub.α F.sub.β whereinthe sum of α and β is sufficient to satisfy the otherwise unsatisfiedvalencies of the C, N, and O atoms; ##STR40## R¹ is hydrogen or C₁₋₁₀alkyl; c is an integer ranging from 0 to 4; and T⁵ is an organic moietyof the formula C₁₋₂₅ N₀₋₉ O₀₋₉ H.sub.α F.sub.β wherein the sum of ax andD is sufficient to satisfy the otherwise unsatisfied valencies of the C,N, and O atoms; and T⁵ includes a tertiary or quaternary amine or anorganic acid; and m is an integer ranging from 0-49 where when m isgreater than 1, then G, c, Amide, R¹ and T⁴ are independently selected.67. The method of claim 20 wherein the tagged nucleic acid probe is acompound having a mass of at least 250 daltons of the formula:

    T.sup.ms -L-X

wherein T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; Lis an organic group which allows a T^(ms) -containing moiety to becleaved from the remainder of the compound, wherein the T^(ms)-containing moiety comprises a functional group which supports a singleionized charge state when the compound is subjected to mass spectrometryand is selected from tertiary amine, quaternary amine and organic acid;X comprises a nucleic acid probe; the compound also having the formula:##STR41## wherein G is (CH₂)₁₋₆ wherein a hydrogen on one and only oneof the CH₂ groups is replaced with-(CH₂)_(c) -Amide-T⁴ ; T² and T⁴ areorganic moieties of the formula C₁₋₂₅ N₀₋₉ O₀₋₉ H.sub.α F.sub.β whereinthe sum of α and β is sufficient to satisfy the otherwise unsatisfiedvalencies of the C, N, and O atoms; ##STR42## R¹ is hydrogen or C₁₋₁₀alkyl; c is an integer ranging from 0 to 4; and T⁵ is an organic moietyof the formula C₁₋₂₅ N₀₋₉ O₀₋₉ H.sub.α F.sub.β wherein the sum of α andβ is sufficient to satisfy the otherwise unsatisfied valencies of the C,N, and O atoms; and T⁵ includes a tertiary or quaternary amine or anorganic acid; and m is an integer ranging from 0-49 where when m isgreater than 1, then G, c, Amide, R¹ and T⁴ are independently selected.68. A composition comprising a pair of compounds of the formula:

    T.sup.ms -L-MOI

wherein, T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; Lis an organic group which allows a T^(ms) -containing moiety to becleaved from the remainder of the compound, wherein the T^(ms)-containing moiety comprises a functional group which supports a singleionized charge state when the compound is subjected to mass spectrometryand is selected from tertiary amine, quaternary amine and organic acid;MOI is a nucleic acid fragment wherein L is conjugated to MOI at otherthan the 3' end of the MOI; and the compounds of the pair havenon-identical T^(ms) groups, and have identical sequences except at onebase position where the bases are non-identical.
 69. A compositioncomprising a pair of compounds of the formula:

    T.sup.ms -L-MOI

wherein, T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; Lis an organic group which allows a T^(ms) -containing moiety to becleaved from the remainder of the compound, wherein the T^(ms)-containing moiety comprises a functional group which supports a singleionized charge state when the compound is subjected to mass spectrometryand is selected from tertiary amine, quaternary amine and organic acid;MOI is a nucleic acid fragment wherein L is conjugated to MOI at otherthan the 3' end of the MOI; and the compounds of the pair havenon-identical T^(ms) groups, and have identical sequences except at twobase position where the bases are non-identical.
 70. A compositionaccording to claim any of claims 68 or 69, comprising a plurality of thepairs.
 71. A composition according to any of claims 68 or 69, comprisinga plurality of the pairs, and an equal plurality of non-identicalnucleic acids immobilized on a solid support, wherein each member of theplurality of nucleic acids has a base sequence that is exactlycomplementary to one member of each of the pairs.
 72. The methodaccording to claim 1 wherein said first and second members are nucleicacid molecules and differ by at least a single base-pair mismatch.